I. A Plant Growth Inhibitor and Plant Growth Inhibition. II. Extensibility of Cell Wall Material in Indole-3-Acetic Acid Citation Stewart, William Sheldon (1939) I. A Plant Growth Inhibitor and Plant Growth Inhibition. II. Extensibility of Cell Wall Material in Indole-3-Acetic Acid. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/jgkt-9a09. https://resolver.caltech.edu/CaltechTHESIS:12182024-183614320 Abstract No abstract. Item Type: Thesis (Dissertation (Ph.D.)) Subject Keywords: (Biology) Degree Grantor: California Institute of Technology Division: Biology Major Option: Biology Thesis Availability: Public (worldwide access) Research Advisor(s): Unknown, Unknown Thesis Committee: Unknown, Unknown Defense Date: 1 January 1939 Record Number: CaltechTHESIS:12182024-183614320 Persistent URL: https://resolver.caltech.edu/CaltechTHESIS:12182024-183614320 DOI: 10.7907/jgkt-9a09 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 16940 Collection: CaltechTHESIS Deposited By: Benjamin Perez Deposited On: 18 Dec 2024 19:23 Last Modified: 18 Dec 2024 19:44 Thesis Files PDF - Final Version See Usage Policy. 39MB Repository Staff Only: item control page

A 1Thesis t,ubm.:t tted in Two Perts: 1. A Plant Growth Inhibitor end Plant Growth Inhibition . 2. Extensibility of Cell Wa ll Materie.1 i n lndole-3-acetic Aci d . by William t,heldon Stewart., In Pt:artia.1 7t1lfillr11ent of the Hequ irernents f'or the Degree of Doctor of Philosophy. California Institute of Technology , Pa s adena, California . 1939. i'.', um:£11:u•y of a Thesis f.?ubmi tted in 1'wo I-arts: 1. A Plant Growth Inhibitor and Plant Growth Inhibition. 2. Extensibility of Cell Wall :1h, terial in Indole-3-Acetic Acid. 1. A Plan t Growth Inhi b itor and Plant Growth inhibi t ion. A non•toxic ether ex trt1.c t able s u bst.e.nce, capable of' inhlbi ting plant cell elonga tion growth , was f ound in the cotyledons ru1d leoves o f varis)ua herbaceous plants. He.d ish coty ledons had the h i r;hest concentration. of this 11 inhibitor 11 substance of any of t he sources investica ted. \".ihen the inh.ib i tor was applied at the top of an fwena plant coleoptile, the inh i b it i on was manifested as a pos itive courature w:i.thin 2½ hours. Between 3 and 13 de grees., the curvature was line arly pr()portional to the Ln:1.:ibi tor concentration. rl:'nus by :11.c nsurinc: the cur v ~ture it was po ;:; sible to e.na.l y ze quanti r; a ... tively for the inri.ib i tor. 'I'he inhH: :itor was fo und to be stored in the ra:-.5.sh seed, syn t hesized in the plant, and ta d i sappear with leaf growth. Chemical investi g ations indicate t h0 t t h e inhib itor is en e s t er of ind ole ncetic acid with so:ne as yet undetermined s :.1bs tance. In a purified form this ester is readily hydrolysed in water to form auxin. 'l1he aroount of hydrolysis is decreased by t he addition o f various substances or t he presence Df im• pur:tties in the extract. Growth meas urements showed that the positive curvatures could be accounted for by- the ,neasured amount of inh:tbi tion. The movement of inhib itor throu ,..,..h Avena eo leoptiles is non,101.nr. This fact was used to separate c on ta.minatinc auxin from c:m.1de inhibitor extract by collectinr:: t he inhib itor mov ing tnro~~h the coleopti le acropetally. - Au. x in can not be transpot>ted :i.n tb. is direction as i t is only passed throu (':h the coleoptile be.sipeta1ly. 'Ihe velocity of i nhi b itor transport i n the coleoptile is a pproz. lms.tely 11 mm., / hour. Diffusion coefficient determinations indicate that hydrolysis 0f .l:r1hl.b i tor could a.cc :)U!'l t for the att."tin ( probt3bly inclo le r: :. cetic Heid) diffusing fro ,1 radish c oty ledons. The determine.tions also indicate t hat tht➔- t the molecular weieht of the inhi bitor may be :.e tween 100 and 175 2. Lxte n si.hil:t ty of Cell Well G1a terial in Indole-3-acetic Acid. l r t:tficial silk was found to he.ve an. increased e.x tensibili ty, compared to water controls, in certain organic acids 1m,)wn not to b e c·r~:rwth subs t.an.ces as well as in • 2)" indole ace ti c ac :l.d. • Onion roots were found to have an increased extensibility in .2~ indole acetic hc i d and in .2% acetic acid. :C.:xperimente of Hobbins and Ja.c kson were repee.tod and found t~ p ive n o conclusive evi dence as to the e f fect of indole 0.cotic ac:l.d on t h e extensibility of stem a.nd root cell wall materials. TABLE Part 1. OF' A Plant Growth I nhibitor• and Plant Growth Inh:l.bi tion. Chapter I. A Plant Growth Inhi bitor. A. Introduction . B. :2;arly work on s ,1bs t ances causing })OSi t ive c. D. E. rrrowth cnrv 2 t ures in /, vena cole optiles. 2. Recen t work on s ubstances causing positive growth curvatures in Avena col0optiles. 3. Defini ti on and fac t ors caus i ng positiv e growth curvatures in Avena coleoptiles. 6• Discovery of a tru.e growth inhib iting sub stance in r• adis h coty ledons,. • a. Chapter c. l, II. The Bio--a.ssay for Growth lnhib l tor. 10 Cn:i_;•v a ture r a te of Avena coleo pt iles t o i nhib i tor. 11 Relation betwc n positive curvature and c 0nc3n tration of inhlbi tor. 12 Positive curv e, t ure rates of Avena coleoptiles of different auxin sensitivity. Cha pter I I I. 15 Distribu tion of Inhib itor. A. Sources of inh i b i t or. 21 B. Distr:l.bution of i nhibitor in the radish plmnt. 25 Chapter rv. Chemical Properties of the Inhi bitor. A. Solven ts for i n hib i t or e xtrect i on. 26 B. Hydrolysis of :l.nhi Li t or t o y ield a ux i n . 2? c. Id e nt :L f :i..eat ion of t h e a uxin fro m inh i bi'-, or. 27 ii. Chapter v. Growth I nhibitor an d Growth .. A. Growth effect of inhibitor on Avena c oleoptiles. 31 B. Observed positive curvature explained on a basis of n1easured growth inhibition. 35 Growth effect of inhibitor on tiss u e sections of Avena and radish. 35 c. Chapter VI. A. B. c. Inhibitor 'l'ransport 'l ':tironc:h Avena. and Redish Sections. Non-polar movement of inhibitor in Avena coleoptiles and 1~adish hypocotyls • 38 Chronolog ical record of movement of inhibitor through sect:Luns of Avena coleop t :tles and radish hypocotylst- 41 Auxin contami nation of inhibitor extract and its removal bv the 0 Inverse TrensDort" purification method. 43 Action o.f f,vena test plan ts to inhibitor conta..11inated with a ,Lxin, 45 y D. E. F. G. ~ Curvature rates of Avena coleoptiles to dilutions of inhibitor, 47 l1rs.nsport of purified inhihi tor in Avena coleoptile secti ,na. 50 1 Transport rste of purified inhib itor in Avena coleoptiles. H. 52 Transport of purified inhibitor in radish hypocotyl sections. Chapter VII., 54 Physi tJ log ical Role of Inhibitor. A. Inhibitor does not inhib it lmd growth. 57 B. Inhibitor is not active in mesopbyll g row:th. 58 c. Inhibitor inhib its root g row t h. 59 D. Indole ,acetic acid does not inhlb i t root .rrowth by causing an accur.iu la tion. of inhibitor. 61 iii. E. Ir1hll1i tor is riot s.ctiv·e ir1 root for rnatio·n . 62 f<' ., Inhibitor inhibits EJeed g ermin$.tion.. 63 G.. Growth inb.ibi tlon 1J y lnh ibi tor in tissues othe:r than the Avena eoleoptile. 65 Inhibitor does not af f ect the phototropic or r;eotropic response of Avena coleoptiles. 66 I. .tnhibl tor aoes not perme.nen tly stop growth. 68 J,_ Aux.in-like action of inh:tbitor in t he pea stem growth test. 69 H. K. Possib:i.11 ty of i n hib itor acting l:lke an auxin precursor. L. M. Effect of inhibitor on a u.xin prodlie tion in Avena coleoptile tips a.na radish cotyledons. 73 Conclnaions. '75 Chapter VIII. A. Mechanism of the Inhib iting Action . Effect of inhibitor on food factor distri?6 i:-iu tion • B. Action of lipase preparations on purific-,d .inhibitor. c. n. r.:i, 79 Prevention of inhibitor pydrolysis by impurities in t h (~ extrt<ct,. 31 Conclusions. 82 Chapter I X. .l.J . 71 Discussion and Conclusions. Discussion. 34 Conclusions. 87 Bi ::: liog re.ph7 . 39 iV• Part 2. Ex 1:, en sib ili ty of Cell Vi a.11 MBterial in Indolc - 3 - ec c ti c Acid. c. n. In trodnc tio11 . 1 :E xp ,r imen ts an rJ rerrn l t s . 2 Dis c ussion . 8 Ei b 11 ogr a pby . 10 11 Pa.rt 1. A Plant Growth Inhibitor and Plan t Growth Inhibition . 1. Chapter I . A.. A Plant Growth Inhi .i tor I n trod uction .. '.Pwo types of' r;r owth inhibition are ::- enera.11:y con sidered ir1 p lant phys::i.D l o:::YtUscuss ed is th1:1.t c oncerned Clne first and nost wid ely w j_ t h the :iie.intenenco . ,f the :~ orme r~ t sta t e of plan t tissi...1e s; seed g erminati on , b' d iDhibi ti.on ; e tc . g_ F,;.,d '.Lntiib i ti o:n i s ofter; spoken Df' a s 0correlation " p"r1en o~cn on :Ln vievi ,J f t t.1e fact t ho t the non - deve l opment of t he 1 : - teral bu d s is d eter mined :~ y t h e prese nce of t he ter ·n:-_n e.1 bud .. It ls c ommon l J a g r- c .;: d t t.nt the termin al b 11.d maintain s its ,: o::ninnnce over t he l a ter a l b uds y its pro du ction of auxin ; however, the me c han ism b y whi.ch a.uxin c s.uses t 1-!i f, L ;h ihi tlon to take p h ;.ce i s still Hn open q ,:(estion . The nun1erou s theories ab ou t thi s Clat ter ar e well summarized by VHn Overb ·e ek ( 193 3 ) . The second type of c r owth :tnhl bi t lon , h owev er, is n ot concerned with t.10 maintenance of a d or mant state as in ud :;_nh:1.bttion , but is a c tua lly c oncer ned with. t h e :in terfer en ce of l;he e long ation c r owt~ of th.c cell. It is this k i nd of inhibition with wh i ch we ar e con cerned . 1:f a c ro wth promoting sub stance is a pplied :i.n a l ~}b ::1gar b lock , siz e 1 x 2 x 2 mm.. , n :i. a 4:enally to the cut s i rface of t he t o p o f d e c a pit at ed Au e ~ 2 coleoptile , 2. and if the substance is c 1-.:1pab le of' b einc; t r ansported dovm the co l eopt i l e , t he c oleopti le wi l l curve in a direc- tion away fro :.-ri. t he ap;ar b lo ck . 'I>his is caused by a g:r•ea. t - er growth of t he treated s ide t han of the untreated . i s kno1w1'n as s. n e g ative Av e n a c urva ture, This I f , however, the su:J ~; t a.nee cfl.nse s. an inhib ition of g rowth , t hen the un• t r eated side wil l gr ow m.ore than t h e tre a t ed and the cur ... vature wil l b e toward the ag ar !)loc k . a posi t i v e c urvature . r.rhis is lmovm a s {F or i l h:.strations of posL.,. ive and neg ati ve curvatur e s s ee fi gur e 1,.) Thu s it is clear that in con s idering s tilb st ances c ausing an i nhib ition of cell e l ongsd;ion gr owth , one rnus t; consi der pos i t:l ve J..vena. eurvatur e s .. B. 1'. a rly wor k on subs tan.ces caus ing p osi t ive gr owth c ur·v atur e s in Av ena coleopti l es . A summary of pr evi ous wo1~k on pos i t i ve f1v ena c ur ... vatures is f oun d i n Phy t ohor mones , ( Wen t .'.c 'I11limann , 1937) . Her e it is pointed out t h at t he positive c ur vatu.res ob tained 1:,y r:t 0r k ( 19 21), Nelson {19 24), and Eeuber t (19 24 ) , wer e probe.b l y not c au sed by grow th inhibiting substances a s they c l ai med t u t we re c au sed by t h e unequa l r estimpt ion of au.x in pr ,o uc t ion of the Avene. s tu.mp ( 0 phys io1og ic a l tip n ) a.f t er t h e norrnR.l ti :p ht\d b een remov ed . ..'U so b oth Star k and ~: eu ...,ert f ~1i l ed to g i ve the magnitud e and cur ... :> -1- , ,-4 ./J,-f ·------i (1) " ' 0 ,r. 0 I u I + t d ■ ~ . II I l,:l 'l) :> I) 1H (I) f-, .p ) :> ,.., $--, 0 ➔ -I 3. vatur•e re.te of t heir test plants fo r t h e substances inv c sti :·: nted . For these reasons it i s c1i ffi c u l t to draw any cnnclus i ons from their data. . Gorte,r ( 19 2'7 ) firs t found a relations hip to exist between positive curvatur e s (of rath er slif~ht :aagni tude) 1 and the ' phys io log ice.l tip,. re;:~enere.t i on ; althoug h s h e did not re e. lize tha t the positive curv e tu.res were c au sed b y an unequ al auxin production at t his reg enerated tip, s he did find t hat t hey a lways a ppeared after t he two and a h e.lf hours w·'.1ic h are neces s ary for reg en eration of e. 11 physi olor, ical tip 0 • Si nce positive curvatures never ap- peare d in les s t h an this time, and since sub stances claimed to be c;rowth inhibitors by previous investig ators g ave t he sa:11e positive ctu•va t u.res e.s ple.in ag ar , s h e ca:ne t o the c onclusion thv. t the tip r egenerat i on was the c ause o:r. t l1e :? ositive c tirve.tures . In concludihg her paper she says: ... "Su.rnm.arizing one esn t herefore say that at pre sent on l y grow th acc elerating sub stanc e s have been found . Only when one will f ind a substance t h il t wi l l c&u se a posi t ive c urvature within two a.nd a half hours may one speak of e. growth. i nhib iting substance . " c. Re cent work on sub stances c ausing po si t ive growth curv a t ur es in Avena coleoptiles . 4. Recently Skoog (1936) has found tha.t a block of pure agar on the top of a deca pitated Avena coleoptile ac t ually inhibits the formation of auxin in t he regeaere.ting tip. This he.s t he result t hat when pure ag ar alone 1s ple.ced on onf'l aide of the Avena. test plants, the production of suxin after two and a half hours will be leas on t he side of the plant below the agar block t her1 on the side without it. That side wi').l hence have a decreased growth rate end positive curvatures will thus appear. This completely ex plains the r elat.1 onsh1p suggested by Gorter between the onset of tip regeneration and the appenrance of positive curvatu.rea. Meyer (1936) has re ported poait1ve Avena curvatures from plant extracts prepared by dehyd,rating the plant mat... er1al with alcohol, grinding tt to a powder in a 111ortar and extre.oting it with lanolin. The lanolin extract waa then applied to Avena eoleoptiles e.eeo:rding to tl1.e technique of Laibach (1933) and the curvature measured after 24 hours •. ·w1th this method i t is difficult to see how any po.si t ive curvatures could be caused by the tip regeneration effe ct of Gorter e.s in this method the plant is not decapitated. Except f'or nasturtium cotyledon extract the s ubstances were only i nhibiting e.t higher eoneentra.t1 ons. At lower eoncentrations a negative curvature res ulted. This would i ndicate a t l.)xie acti on at t he hie;her concentrations. An ether extre.ct of t he !~ asturtlum cotyledons 5. e.cc ordin ;~ to t _1e ::1et.hod used fo r r adi s h c otyl eaonri s howed the presen ce of ,growth .inhFii tor. ( See Ch ap t er I l l . ) Skoog (1936) found t hat indole ethyl amine and also t r yptoph an e e.re c apable of c ausing sligh t positive c urve.• t ures Vlith i n t he f irs t two hours when t ested on des eeded _plan t s, but t h.f t t hese posi t ive curv atures soon t here.af ter change in t o ne g at ive on es ; prob ab l y upon the i r tran s f orma- t ion in t he pl an t, i nto grow t h pr omo t ing s ubst ances. They are t hus certainly no t 'to b e c onsidered es growth inh ibit• ine; sub s tances. Czaja (1934) has re ported t h nt tissues rich in tannin• for example ca.11,ls t issue of Po pulus, are ab le in some c a ses t o di f fu se out e. grow t h inhib i t'fmg substance. He also found t ha t tann ic acid an d g a l lic acid of a 1 1; concentrat ion are likewi se cap.able of causing pos i tive c ur•v atures. These curva tur e s he at t rib utes to t he s ctual shr i nkag e of the c oleopt;ile cells on t h e t r ea ted si de so tha t t hey act ually b ecome a.:10rter than those on the untr eated pa.rt . He ob t ai ned a ma.,"ti mu:m c urva ture of t wenty de grees i n t hree hours . Upon. r e peating h is experiment using l j{ soluti ons of t ann ic anc1 gallic ac i ds I was unab le t o ob ta in any ) O Si t ive c ur vature s whatsoever. rn1ile t his r,1ay have b een due t o t he fact tha t C2' a ja p1•ep ared 111.s as o.r test b locks by soaking them f or 24 hours in t h e s olut i ons and I pr ,~,pare cl m.ine by taki ng t he solu tions up in melted aga.r so t hnt t he fl nal 6. concen tration was the same as Czaja's, it seems unlikely that this s light differe nce in procedure s hou l d make any difference in t :~e final acti on of the sub stances themselves on the test plants. Brecht (1936) has s h ovm. t hat ac etic aci d in l anoli n pa s t e when applied dir ectl y t o intact .!:.", ena co l e op t ile s 7 is capabl0 of caus ing posi t ive curvatures . tures, however , are pr ec e ded T}:1ese c urva- '":Jy neg ati ve curva tures and are t he:ns e lve,s '"' c knowledg ed t o b e t h e r e s ult of an i njury effect of the G? cetic acid . Vi ehman (1938 ) has recently f ound that l anolin pas te s of acetic and of formic acid in c oncentra t i ;ns of f r om l . 0625 M to .25M when.applied to t h e top of a.,(cm. long piece of hypoc otyl of .i. Ielianthus or Lupinus c a uses a s ub• stan ce t o d iffus e out wh ic I1 i s capab l e of elici t i n t: : posi• t 1ve Avena C\J.rva t ur e s. Th ea e curvatures are .: e ver ini- tiated in less t han two hours. D. Definition and fact ors causing positive growth curv atures i n Avena coleoptiles . The statemen t :ms.y b e s nfe l y ;nade t h at whenever a posit i v c curvature occurs in en Av ens plan t t h.er e ha:,1 been a rela t i.ve inhibition of g rowth . Sinc e t hi s inh ibiti on i s t h e resu lt of a d is tur b an ce of tl1:e nor mal increa s e in cell valume it is de sireable t o c onsid.e r for & moment the 7. f actors govern ing t h is process t h at c ould b e affected to g i ve e. deor e esed. growth rate. For ou.r purposes gr owth may be defined as an irrev ers ib le i ncrease in cell volume. This gr owth may be broug ht about e i t .1er by: 1. we..ter upt ake; 2. r:m in• crease of t he cell cons t ituents .mainly b y cell wall f orma ti on; or 3. both pr oc esses w -·,k ins t og eth er . The last ease is t lie one t h e.t occurs bu t in it water \lPtak e is the major factor . {Phy toh ormones, 1937.) The ab ility ,: if a cell to take up water may be said to b e t h e res u l t of t h e di f f erence t:i e tween t he o sm,ot ic pressure of t h e eell conten ts and their tendency t o take u p water , and t he oppo s ite act i on {tur g or pressure) of the c e ll wall whic h s urrounds the cel l cont ents and ex- erts a compressing for c e on them.• or tends to caus e them. to lose WFt er. Tb.is d i f f erence betwee n t he t e n dency of t h e cell conten t s to take u p v1e.ter and t h e opposing tendency on the p art of t he cell wall, has b ee n c' e.t'1ned 1,y Ur sprung and Blum ( 1924) as the ii sue tion F'orce n • I t is then due to t he suction force tha t e. cell is nb le to in ... crease its water con tent and hence to e;r ow . A dec r ease either in s uction f orce or in a va i lable water will t hus decrease the amount of growth of t he cell. 'l'h e auction f orce may b e decre as ed b y lovrnring t h e osmotic pre ssure , increasing t he t ur g o:t, pressure {compressing force) on the cell contents, i n ,iu.ry, or death. The water avail- 8. able t o the cell may be decreased by i nterrupting the nor r.1e.l water supply ei thcr b: i nterfering with the trans-· 0 port of t he water in the c onducting sys t ern or by a pplying hyperton1c s oluti ons.,. Any one, or c omb ination of t hese f actors would thus be ca pable of c&us ing s. :rela t ive gr owth inh ibition and hence a re still ting posi.ti ve vurva- tu:re .. In view of the e.bove fact s, we ti1ey now ~ay thn t sub• s t ances claim.ed t o be inhibitors of the cell gr owth proces s i tself must act either by : i ng the osmo tic pressure., pr es sure . 1. 01" l. By r e ve1~si bly lower- 2. by :inereaaine; t he t u.rgor rub s ta.'1:ces which cause posi t:i. ve cu::•va tures by : exosmosis, as for o.xumplc the salts investigated by I<: isser and !::ieor, ( 1934), or 2. by in juring the cell so t hat it i s no longer c apnble of growth, e.s shown by Brecht ( 1936) f or• acetic acid are thus n o t to b e cons idered as ninhibitors" of the growth process. E,. Discover y of a true growth inhibiting substance in radish cotyledons. An e ther extr action of radish cotyledons a.ceording to the metho : 1: of Van Ovor b eek (1933) fo r a :. u:in fai led to s how any au xin but inste ad s hovred t he presence of an i n• hibi ting s ub stance., TI-u s wa s manifes t ed after 90 minutes as a positive curva ture i n t he stands.rd Avena test 9. ( P:b..ytoh orm.ones-, 193? ) . Curvatures ob te.ined with this extract may be seen in figure 1. The extracted cotyledons were from radish plants of the strain "French B:reakfastn. The pl ants were seven days old and had been grown in the open in loamy soil. dons were used . Five grams fresh weight of cotyle• At the sa:m.e ti:ne the.t these posi t ive cur- v:;tures were f ound Mr . ·wm. Bergren of t hese laborateries , repor ted s in:lil 1;1r c urvatures from ether extracts c otyledons , i:; f re.C:ish A pi-• e li:ninary note describing t hese proper- t ies hes b een pub lished . ( 2.tewart, Redema..nn , and Eerc;r en , 1939) . From the fo regoing discussion we have seen t hat a t rue e;rov.rth inhibitor niust be ab le to cau se po sitive Aven a coleoptile curve.tur•es in less than two hou..,'"s , snd ~nu s t neither oauae the cells to pe rms nently l ose their growth nbili ty, nor to become plas:nolysed ., Preldniin.a ry observa- t ions showed thet the rs.dish cotyledon extract s a tisfied these requi rements . Heretofore no substance had been :'ound whicl1 so completely fulfil l ed the cr1 teria for a true growth inhibitor. It became the object of this work to in• vesti " o.te the properties of t his cotyledon extrac t . In s ub sequent discussion r 1;fere:nce made to growth inhibitor will refer to t hi s subs tance . 10. Chapter II . The Bio- assay for Growth Inhibi tor . I n Chapter I i t was seen that it was possib le to ob- tain from radis h cotyledons, by the auxin e ther "Jxtraction m.ethod, a s ub s tanee t hc.t caused positive cur vatare a in lwena plants. It we.s next of inter es t to know if t he s tan.. da.rd .Avena. test ,perhaps with ,i1odifice.tions , could be u sed as a bio- e.saay for t his i nhi bitor s ubs tance . this point i t was necessary to know: 1.} To s e tt le The op ti.m.al time after appl i c ation of the inhibitor to measur e the posi t ive curv a ture; 2.} The c ur vature ranee which wa s 11n es.r.l y prop,: :>rtional to t he con cen t:ra t ion of the i n r_1ibi t or , an d 3.) The most reac tive type of t est pl ants to use . The answers t o these questions were sough t by the experiments describ ed below. Thr oughout thi s t h es i s the nw.terials and methods ri.s ed are thos e which are so e ompletely described i n "Phy tohormonestf (Went and Thinumn , 1937) t ha t repeated wi ll no t b e g iven here. For exa.mple , i t enumer a ., ion of them 1 t o b e under- s to od that t he Avena test plants were grown f r om t he standard t~ iec; esha.fer strain of s eed according to t he r i g i d tech- nique out lined in the a foreme n tioned monograph . Inhib itor was always pre pared ac cording to the me thod des cr ibed i n Ch apter• I and a pplied to t he Avena plan ts i n l ½}t ag ar b loc ks unl es r:; other wise s pecifically noted . 11. A. Cu.rvs.ture r ate of .Avena c oleoptiles to inhibitor. To determine the optimal time for meas uring t he angle of curvature a "Pho tokymographfl te st was made according to the method given by Schneider and Went (1938) and improved by Went and White (1939). In making tbia test the growth ,aovemen t of the Avena test c oleoptile ,during e. g iven per• iod of tim.e , is recorded photogr aphically by mean s of a shadou c as t on & revolving drum o.f photographic paper., 1i1;1:; .s ha.dow cast is not t h a t of the coleopt11e itself bu t is that of e. ver y fine :(No., 36 , .012 mm. diameter) silver wire inserted in the primary leaf. The primary leaf is pr e- vi ously broken loose as is the procedure in the Avena. test . Tb.is prevents it from furt her growth and keeps it from lift .... ing the. ggar :Jloeks up f rom the cut top of t he coleoptile . Inser ting the si lver wire and thus ne:."ttending " in ef fect t h e coleoptile has two e.dve.ntages. First, it keeps the c oleopti le its-elf in t he dar k and allows on l y the si lver wir·e to intereept the beam of l ight thus preventing photot ropic curvatures of the c oleoptlle. and sec ondl y it amplifie.s the curvature. At s1nall ,m.gles a c urvature of 1 moves t he shndow on t he ~um about 1 I!'h1l• 0 This whole mac hine :ts kept in the r egul ar Avena t e sting de;rk ro om at con stan t humi dity and te mper ature. T'he resu l ts of suc h a t ti.s t f or the inhib i ·:~'-i r are shovirn in :flgure 2. ( The c urvature values, the or dinate 16 m. ;: 12 ::, <...) -~ 8 i,, QJ :z 4 Growth Inhibitor 24 m. Curvature rate of Avena plants upon application of: (a) .05 mg/liter, indole-3-acetic acid, and (b) growth inhibitor. (Ordinate values given as devia..i. _ tion of the extended coleoptile from the ~~rt~cal position.) = Concentrat,on I!,elation between positive curvature of Avena plants and two-fold dilutions of inhibitor substance. F ii::,ure 4 . 12. axis for this exper·i:men t , Imd all s ub sequent photo~.y mograph tests , wi ll be gi ven in actual mm. deflection of the st).,.. v e:r wire or nextended c ol eopti l ,e t:tp" •) It i s obs erved f'ro~ this data t h.e..t for t h e i nhibitor ~ a neg ative curvature is initiated Wh i c h l"' a pidl y ch..anges betweer:i the first and second hour i nto a positive c urva ture a.nd ~~aches a.· ma..·•,d.,. mum in ab out t hr·ee hou1~s. I t is i ntePe sting to n ote that the positive curvatur e is dit'f'eren t f :rom t he nega tive curvature caused by pure aux.in a.lone,; contr ol l?iID This is shown by the m!ld-e a t the sa.m.e time as the i nhibitor tes t bat by us :i.ng a 13rowth. p1"omo ting s ubstance ., ind.o 1e ... 3 ...e;c e tic eeid , .05 mgij per li t er... Each point on t he gr aph is the a ver 8.g e of twe1--ve Avena t est pl ants . Tb.is expe r iraen t ha s been repe ated many times s 1ow:tng the same ~'time-positive ... An o ther experiment of tJ:-i.e same natur e was porform.ed e-:iwept i n this c e.se the 1nhi'b'.L tor was a ppli ed un:'i.1Hcera11y i n lanolin p a s -.-; e at; t he t,ip of intact plants" 1tlle silver wire t o 11 ez.:tena.n t he coleopt:tle was :i. n s er ted through the tip of tb.e pla:nt and dmm into -the primary le af ~ I t is se en in figure 3 . t h.a t the pos i tiv e cu:rvatu.re ra t G l s the same he:re t;i.s in the t es t us :i.ng inhib i to1· a pplied in P-<f;o.r blocks a.l t h ou,eh t _1e in1 tie.1 n cies.t;i ve e urv-a t ure i .s absent. Thi$ p oin t will be d i scm1:.s ed :Ln Ch&.pter VI , Fig. ~ Heaction ra t e of Avena pla nts to inhibi to r applied in lano lin t o inta~t pl~ntso n eq,u~ls nur..ber of 1:->lant s avero.,r,ed for each poin t . • Negati ve Curvature 2 ~~--~~----~--+----trcomn t ro 1, lan C, l i n ur: ly . n = 4. 10 20 Positive Curvi:.l. ture ( in mr11. ) 3 plus inhi b t :or . n = 8, 40 13. From both of ·these expeviments it is elearly seen ths,t after a. 150 minu tes the <H1rvature is st i ll proceding a t the ma1timum rate, but that soon thereafter 1 t ber:;ins to decrease rapidly. From tbis date. then the time foI' measur -. ing the ang le of positive cu:t?-vature wa.s set at 150 minu tes from the time of applic ation of' the inhibitor~ B·. Relation be twee· positive curvature and con ... eent:ra tion of' inhibitor ·. To an swer the second question it 'W&1 nec e ssary to determine the relation between the c oncentr ation of t'le i nhibitor and the magnitude of positive curvature . This was don e by the following experiment: A concentration series of an ac t ive :tnhibi tor· e" - tract was prepared and 48 .Avena pl ants ua e d. to d eternrl.ne the degrees of positive curve.tu.re et each eoncentration value . The standard : vena tech.l'lique we.s used. except t h at t he curvatures were meas ired after 150 minut es fro m the time of applic-ati Gn of t he inhibitor :i..nsteA.d of' a.fter 90 min--. utes as is customary when one is testing e;l?owth pr omoting su.b s tances. 'l'Jne rea ul ta ar e seen in .figure 4 . Th is g re,ph shows that positive curva tures between thr'ee and thi:r t een degrees a.re linearly propo:t> ti onnl to the amount of inhibi -tor . 'rhis exper imen t has been repeated me.ny timec with simi l ar res ults~ See Chapter IV . 14. The neon centration ... <.mrvature 0 re16'.tionship was a.1so investigated using a concentration series of :i.nhibitor in le.nolin. The lanolin was applied 1..1..nilaterally on intact plants over a 5 mm. strip,, i n one case at the tip and. in another ease at the mi d dl.e , of t!:le coleoptile, Applying a lanolin p-a.ste at the bottom of thr: coleopti le did not cause any positive curvature,. The positive enrvatures :resulting f rom the applications a t the t i p and middle were measured after two hours. Measurements of the curvatures after four hours showed there had been no inere~se in t he sensitivity of the teat . At the same time thcst the lano lin was applied , a sim:i.l a:r tes t u s:ing the same amounts of inh.ibi ... t or in ag ar b loc ks was r un .. ms.rized in fi gure 5 . All of these resu lts a:r.e sum... ( Pic tur es of curved plants may be aeem i n fig . 22,. - Chapt .•. VI.) i t may be seen that the method 0 . appl yi ng inb.ibitor in lano lin at the middle of the coleo:ptile gives the gr eatest rang e over which a lin ... ear. pr oportionality exists ·between curvature and eoncen ... t:ration . The sam.e range exists Vih en the l anolin with inhi ... bitor is applied at the tip ; however, i n t his case t he magni tude of c urv ature, is considerably le,ss . It is inter• esting to see t lua t when the same a:nount of inhib itor is ap.-., plied in a.gar the curvature is line arly propor tional CV,;,! just that r ange of concentration in which the lanolin with inhib i t or is not proportional,. It is also significant U I 15c I, 1 Positive Curvature. 250 I. 3 I .s 1 - tip Response of Avena 1,>la nts to the same an, ) unts of inhibitor applied in lanolin ~t t h e t ip a nd middle of the coleoptile,ani in 1½% aga r at h.e tip . i1:easured d.fter t wo h ours . . Each point is the uver-c,c:; e of ,;,·i elv e _p .l~nts. ·Figure 5.. Concentra tion of Innibitor . .Agar anolin - tip 11. j 15. that t he agar application method is linearly proportional at lower concentrations of j_nh:i.bitor., th.us making it the most sen si tive , of t he three rneth ods fo'.r' t he anal. ... y sis of inhibitor.. The gr ea t e r eurvtiture of t h e i nhj.b i lo.r in lanolin a pplied at the mi dd le ) r' the coleoptile , may be explained: in part by t h e fact that i n this case the ap.p lication is n e f.\1:'er t o the reg ion of' elong ation .. Also when the inhib itor is applied on the outside., the distance between t he i nhibited side of t he coleoptile and t he growing side i s. gre ater and hence gives gr eater curvature., tha.11 when the inhibitor is applied on a part of a transverse sectionas in t he a.gar b lo{~k techn iqu e. c. Positive curv a t ur e rates of .Avena coleoptiles of different a uxin sen sitivity . •.ro determine the type of Avena coleoptile s h owing the hi ghest inhib i t or sensitivity the following ex periment wa s pe :·formed : fw ena p le.n ts were prepared. with four diff~-::t>ent ai.ntin sensitivities . Fir st , ttde seeded.t' ( end osperm r emoved) plants were grown ae cording to the method given b y Skoog (193'7)" This author has shown that such pl an ts e.re more sensitive to a uxin t h an any other type of .Av en a p lant because by re : ·.ov1ng t he endosperm they are unab le to for m as much auxin as t hey other wi s e would . According ly in the 16. Avena te:;st f or a.uxin~vif'here aux i n is app lied , these p lante s how the g re ates·t re sponse. 'I'he second t-y·pe of plant was obtained b y ms.king two decapi tations three hours apar t as in t h e usual Aven a test . Thes e pl e.nt s are less s en sitive to au.~in than the ndeseededn p lants, h owever they a.re ore sensitive than plants which have been dec apita ted only once. ( Phytohor D1ones, 193? ) .• The third group of pl an ts wer e decapitat ed. ju.st t efore ~ppl ying the inhib itor, A fourth group had a 1ui.n a.ppl iec on the tip of the coleop.. tile i n a lano lin pas te of 1/10 1 000 ccm.eentration two hours prtor to the f'irst a ecapita ti on., then after the first d eca p:1.tation they were us e d i mmediately. It was thour;ht tha t ... his added aux in would tend to make t h e plants the least au..x in sensi t:l.ve. '11h es.e four types of plan -ts were then tested on the pho to kymogl:'aph mach ine .with the same concentrati on of inhi bitor in eac h ca se. The technique was the s ame as t ha t previ ously d e s cr · -)ed" By this mesns their reacti on ra.te and magnitude of' curv- a ;m:·e were dete:c>• minedil The res ults of this t est are gi \"'n i n figure 6 . Here it iB cle s rl;y- seen that the plants whic h were decapitated only once , res ponded t he most whi le the plant s showed t he least curvature. 11 de s eededn The twice a ecapita.ted plants we re intermediate :lr1 t h eir gr• owth inhtbi {,ion CU.l''va .... t ure be tween t he once decapitated and t he plants. 11 deseeded 11 'l'he plants whic h had au.."C:ln added in ex ce s s to that naturally present showed even les s gr owth inhibition than Negative Cti,r va t ure Fig. 6 Re~c t i on r at e to inhibitor of test plan t s of varying auxin content. Each poin t average of t welve plan t s. 3. 5. TIME- Hoi..:-.1 s ,, 6mm. 12mm . Positive Curvature -~~ Deseeded ·-------- 18 mmo 24mm . Ajded auxin 3(. m:u1 0 4ecapt, Once d.ecapt , 36 mm... 17. the once deoapi tated ones. This may be e.xplained tn pa.Pt by the f r.tc t that tht) applied auxin .may have e~n1 aed a ;re ... distribution of t he food :factor so that the:re was ta.ore food factor present i n the discarded decapitated tip ot these plants than there •vvas in t he decapitated tip of the untreated pla.nts. ( Went ;; 19:59). ~t'his woul d decrease the amount of sub sequent gr owth in the auxin tr~ated pla:nts more than in the untreated ones !) the relative growth :1.n-,., hib:l. tion of the two sides of t he coleoptile woul d be less and aceordi.:ng l y t he positive curvatnre would be smalle~ •. r.Phis expel."i.ment has been repeated using :purified in.. hibitor ( See Chapter VI . ) with similar res u l ts , figu_re · ?~ Also many comparisons hs.ve been made between "deseeded" ,, a.nd once and twice decapitated plant s and always w.ith re ... sults similar to t ho se :i:•ep ot•ted here . From the ab ove dat a it was thoueht th2.t if the tv1i c e decapitated p l ant s r e s ponded l ess to inhibitor than the onee c1eeapi tated plan ts a "c oncentration..-.pos:t ti ve ctu-vaturet1 g raph of the two types of test pl ~nts would show that the maxirn:Jm positive curvature possib le was gre a ter with the once de esp itated, &nd also that the range of linear proportionali ty would be e;reater wt th them , than the.t found fol" the twice decapi tated p l ants . To test this idea a di lu ... tion s e ries of purified inhibitor was made and the same concentrations of solntions tested on both the o:nee end the twice deca pitat.e d p lants . In fi gure 8a are s hovm the Fig. 7 Reaction rate to purified · inhibitor of test plants of varying auxin content . "n" eo,.uals number of plan t s used at given au&in content. 6 Negative Curvature 3 4 Time in ho u rs . . Deseeded, n= f., Positive Curvature ( in mm.) l Deca.f)it ct.ted n = 6. 24 18 . ex pec t ed r•esults and in fi gure 8b the resu l ts t hat were actually ob tained. It can be seen tha t t hey wer e quite different f:ro m t hose ant:tcipated .. This diff eren ce may be parti a lly expl ained. by the fact that while the inhibitor we. s t hought t o b e auxin f r ee :l t actually was not. _. This is lmown to be the case as t he test p lants were "Ob ... s-erved an hour and a he-lf a:Uter t h e a.ppl i c ~ t ion of thB ~-eaF b loc ks 0-1"1d with the twice decapitated plan ts, at the lower concentrat i ons of inhib itor , t here we r e clearly negative c:1rvatures. 'l"his made it evident why after 2½ hours with the lower c oncentrations 01' inhibitor, the twice decapitated test plants s howe d a smaller positive curvature than t h e once decapi t~ted. The re ason was dne to t h e fact t hat they were mor e fHmsitive to a uxin in the inhibit or t h an were the once decapi tated plants,, thus they curved nega...tively and then had to reg a in t hat a.mount of neg.!1tiv e c urv ature b efore they beg an to s h o•N &ny po si t ive e urva... ture; hence the y •Fina lly showed le s s po sitive c urva ture-. Thi s negative c urvature pvece:ding .. t :1-e posi t ive curvature is further di scussed in Chap ter VI . As stated , however, i t was expec ted that t he maximum t?.ngl e of the bvo kinds of pl ants would be different , That this was not the cas e may be as cribed to the fa ct tha t t h e e:~_perin1en t was done on too small a sc a le_, only t welve test plants being used for each point,, A repetition of it wou l d pro babl y show t ha t the max i mum ang le wa s grea t er for t he once decapi te:ted Jnce deed.pt. Twic e J ecap t, Pos:li.t i "\re Curva ture Concentrat i on · Inhibitor , 1.0 Figure 8a . Scnematic repre s enta t ion of expect e J c oncentr c.1 ti oncurv a ture rel a tionship with once ~nd twice dec ~~ i t a ted pl ants . Pos itive Curvature Twice Jnce 0 0 1/ 4 l Z LO Concentration of Inhibitor. Fi gure 8b . Results obtained fro~ applying punified inhibitor on once and t wice decaf-,itat ed plants . 19 . plants . This has a.lready been s h own by the af orementioned. kyrnograph test., Here t he dj_ff'e·r e nce i n t he curvature ... ~ - hours after the ,. p;:)lication of t he inhi bitor is however 0 not imch gr eater than 4 • From the fo rega ine; exper i ments '.it may be- sai d tha t the plant s that were growing the s lowes t ("de- seeded") re /3 ondea t l1e l e as t to i nhi i tor while those that v1ere growing the fastest responded the most . 'l'his .ma.de it clear thDt the best t ype of plant t o us e in t he bl o- a.s say f or inhi b itor were t hose t hat we1"e only dec a ,;:tt1:1ted onoe .. As menti onec. these we:r•e obte.i n ed by 'f;lsing t h em ittt".fiedit-..tly sfter the first de ... c a.pi tation ,. This 5.s n ot alway s desire1:i.b le ,, however ; bec aus e: fi r st , the time be t wG, en the fi rst and second dec Eipitatio:ns allows an irregular set of ple.nts a c r1a nc e t o b ec ome mor e u.n.iform in s i z0 , ax:i.d , secondly, i. t is n e ce ssar y to s hift the seed pl an t ing to the even ing i n order to have t he pl an ts t he proper size- about noon 36 • hours l ater ~ rtherm.ore the differeno-e s in curv e. t 1re be twe en t he once anc. twice d ec e.pi tated pl ants are for most experiments not great enough to justify the chang e in t he regu lei.r routine of plant pr e,p r..ra tion f or the st"am3.ar-d auxin test . - For t he s e reasons it became t he prac t ice i n the i nhib i tor test , except i n spe cial experimer1 ts , to use Avena test p lants a s the., are grovm for the au..1tin test " Fr om t h e preceertng experiment s we may define the standar d inh i b i to r test as being exactly the sa.rue as the s t a ndard Avena te st except ;l. That posit ive cu r va tu r e s 20 . are measured after 150 minu t e s instead of n egative curva tures after 90 minutes , ~nd; 2,, '1:hat t h e pm:ii t ive curv e tur e is only line arly proportional to tr.1.e concen• .L.. 'C7' 13.1.,l.On f 'C 'c'le ].n!l.:t. " 1 " l)1.• t:.,or •n•e t ween ;;:,\,.,. an u. A 1-z v •d e grees . O . 21. Chapter III . A. Distribu tion of Inhib i to~ $ource s of inhibitor. 1-l avin{._~ d eveloped a. standar dized method of quan ti t a.ti vel y analyzing f or inhibitor sub stance it was next of tnte,y,est to deter mine i t s· occu rrence :)Oth in other s pecies and e s to i ts ac t ual di s tribution within the r nd t sh pl e.nt its e l f . It rrn.s f irst a es ireab le to see if it. was pr e sent in sr.;r a :ln s of ra d:i ~h othe r than the one in w.ht c h i t was ori ... g inally f ound ., i. e. 11 • Fr ench Breakfas t e n I n Tab l e I ar e listed the results of en i nhibitor anal ysis of t he cotyledons of six ~1tr eJ.ns of rad ish .. ':Fhe:rn were eigh t days ol d s~ the time of extr a c tion mi d wer e a l l extracted end te s ted for inhibi tor accor dtng t.o the s tandard method set forth i n Chapt er II . loamy soil . Thes e pl ants 'iH::re g rown in the open , in It is see t from this data t ha t s.11 of the str ains of re.dis h i n vest i g ated conteined the growth inh i ..,. ;'Ji t or, but tha t the s t r ain "F'renc h Br eakf a st " had the most . Sinc e theoccurrenoe f the inh ibitor in the g enus R?-Ph§;:_nu s was we l l estab lis hed. t.he next quest:i.on was a.s to its pT•esence in oth er g enera. of the same fami ly , i . e . t h e ~ c i f erae ,. '1lJ:1:e result s of l\ s urv ey a s d es c ribed above f or the r a dish stI' a ins ., is also shown in 'I'able :c, It is t o b e noted that g rowth inhib i to1.., c an occu.r i1"1 o ther g en era Analysis ot· Co tyledons for Inhibi.tor Su.bstanoe . Material No . of Tes t Plante Degrees Pos . Ourv. per 5 gr . per ao . l½%aga r . Ra.phanus s at iva; Bla()k Spanieh 24 is .a Early Scarlet i4 l7.o Japa.n.ese Al l Sea.eon 24 Fl"enoh Break;fast 24 18. 3 Long Scarlet 24 ll . O White Ieiol~ B4 3.2 Brasa ioa rapa:Ear ly v,n.i te f lat Dutc;;h 24 Brass ioa. nc-1pobra.siJ'10at Swed1sb.... Amer1c.iiUl p\l:rple top ru t a.ba.ga 24 ,,.,- Brase j, c n igl'a.: Os t.r iob plwne nmat~td l2 S. 7 Tropaeo.lum m~ju.$ ·: .Oouble golden yellow n as t--Ul' t 1um . S 4 . 5* * Given as pos it ive curvatu:re eubs tance in 8.,t~n day old eo tyledons. Ext1·act t a ken up in . 5 co . ,li% a.gar. 22. be s i des Raphanus-. At t his ,junc t ur e i t r:-ray b e well to po i nt out that i f both auxin and inhibitor wer e extr acted s i mul t aneou sly from t he same mat eria l the n e t curv atur e m:i.r;ht b e ne1 thel"' e. measure of' e.u.xin , n or of g r owth i nhib i t or. Only i f the in ... hib i t or were in h i g h enough concentr ation to c au se a g r eater posi t ive c urv ature than the n eg ,;, t i ve cur ,·s.tur e c e.u sed by t he auxin m:i.ght; its presence bec ome app er en t i The same conditions woul d be effec t i ve i n nnking extr ac ti ons fo:r auxin de t ernina tions. The same i dea ha s r ec en tly b een a.d... vanced by Goodwin (lS39) who pre sents evidence f r om di ff us i on c onstant dete· minations ( s ee Chapt . IV .) which indi ... ca t es the pr esenc e o~ J):·owth i n htbi ting substances tha t mask the effec t of auxin . Outs i de of t he Cr uc ifer- as, i nhib i t or has b een f ound i n the J.'r•o_p:eGlac e ae and in t h e .P,n ac ar dlac~ese.• turti um., .. - f am:i.ly .rr o~ I n the n as- l uc.ea~, it was found in the e otyle ... don s nine d ay s af ter '1;,;r 1ninst:i.on . This posi t i v e c ur va t ur e s ubs tance f rom the nasturt i um c oty ledon~ was first observed by Meye r ( 1936) . As hi s m.ethod of e.:-ctr ac tion d i ff e r ed f'r om the e t he r extrac t i on us ed for the ra.l'H sh c o t y l e d ons , it wa s c onsidered de sir eable t o make the standar d extr e.c ti on , re s ults of t b.is test are r ec orded i n Tab le L.. The Dr. Haagen ... Smi t (unpub l is hed. ) ha s r e ported the pr esence of inhi b ltor in t he f r uit of t he p0ppe:r t r ee ,_..,. <:? c hi nus mol le, a memger 23. of the fa:ui l w.t Anac a rdiaceae ,. From the l.'¾ Love date i t seems q u l te li ke l y that t h e o <¼l- urremce of :Lnh.ib i tor may be ra ther wlde spread •. might this b e the c~1s e if one believes that its pr es ence c ,.)u l d be O~) s c ur ed by the p:r·e s en ce of auxin . I ndee d wh i l e many ether extr actions of pl ant meteri a l have s :-1own the presence of' ai,uin and n o t of inhib itor, pe rha ps for the r e a son just me:nt:i. oned t he inhibi tor nuiy nl so b e pre s ent , B. Distri bution of inh:Lbitor i n the r"dis h pl.f111 t . It was next of int ere st to irveB ti~ ete the distr ibu~ tion of inhibitor i n t he rad is h p lan t itself . 'l1o d o thi.s r adi s h seed , s t rain aP:ren ch Bree.kf astit , whic h was used throu~hou.t this wor k ) was sown i n f lat b oxes containing l oamy soll , e t inte:rv.a h! of' thr ee t ,Hy a durinc the c 0 ur se of twn and a h alf munths. During was p l anted at one day intervals . 4 .:;;--0 la.st t wo \': eek s it At the e nd of this time the amounts of :1.nhi bi tor wer e d.e ter mtn ed in the co tyl edons r::ind t 1e l eaves of the p l e.n-t . '.Phe l EH.:'l v es wer e extr acted in the orde1~ of' their a ppe aran ce in t wo s , t r acted wa s about 6 mm , long . ~Ph,e smalle st si ze ex .. Except for t h e fi rs t two l eaves , the ter m:l n a l bud was c ons:tde r ed indl ca ti.v e of the araou.nt of inhibitor in the n ext tw o l ee:ves deve l oping f rom it . t r actions of t.he hypocotyl showed no curv atures . Ex- '.Phese de- t erminat;j_ons e.r e g iven in figure 9 as d e gr e es ~;os i t i ve curv a ture pe r fi ve grams f r e s h we:tght of mater i a l when the 8 v, 3nO 40 ° 1. Fi 6 ure 9. -~ \- 1-:r1. le a ves 5 ·n & 6 t h l e <l ves 3. P.ge in weeks. ---------· 7 th hi.;,f-0 ~ .. I 6. 7. b. 9. 10 Inhibi to r iistribution with ~ge, in c ot yledons and leaves of the RaJ ish ~l~nt . E~ch point a ver dge of t we lve te s t pl~nts. Inhibitor C<l l cu l u ~ j pe r 5 g r . fres h wei ~h t ,extrac t t ~k en up in l cc ii; aga r . Le ~tv e '.__; , m,b ,: r e .:i in 1:. l1e o rd 2 r of' tha:ir appearance. I ·-------- 2. \ / · - · / A6 e i n weeKs. ....-·-.......-------·--:----_-_-_-_-_-_ - - - - - - - ·"·/·,,.,.. __ ls t and 2n d le ctv eo" " " --Co t yleJ ,ms '\; -~· .\ c,.i1·vd. ~lire. P o~it ive 60 24. extract is taken up in one cc . of l ½Jt ae;ar . is t h e averag e of twelve test plants. Each point A few determine.-• tions were repeated at a later time and f ound to agree with those g iven here. In fi gure 9 it may also be se en tha t - t 1""1.e amoun t of infl..ib i t or/ill1.e c otyl edon s reac h es a s harp maximum on the eighth d ay and .fr om that t i n1e on the amount declin es until they finally contain no inhibitor but actually have e.uxin . 1.1he d.e-1.relo ping l eav e s from the t erminal primordi a to the taature le.a_f show fl si mi l ar continual decrease in the amount of inhib itor but with no inclici1tion e.t a ny time of an incre/;\se i n inhib itor e.s in the cotylecJons . inhibitor relatiohship in the coty ledons was first deter:mined by I'iir. Red emann. H:ts :res ults were approx i mately the same as t hose presented here. ~hile the graph, figure 9 ., s hows that the concentra• tion per gr am f res h weie,ht of t he l eaves decreas ed with growth it d oes n o t nece ssarily f ollow trw t t he t ota l of inhibi tm• per leaf decreases accordingly, an101.mt A comparison., however, of t he arr10unt of i nhibitor per le af s hows ths t the first two l e £tves of 52 day old plants had enough inhi - bitor per fiv e grams fresh weight. extract taken up in 1 cc. of ag ar, to cause 3½ degrees positive c urva t ure per lee.f, while compar able 69 day ol d leaves s howed a 2½ degree ne3 e.tive curvature . .rhe s econd t wo le aves of 52 d ay 1 old plants had s11f fi cien t inhib i tor to cause 25 degrees 25. _pcH;i tiv e curvature per l eaf while compar able 69 da.y old l eaves only s howed 5 degrees posi t ive c urvature per leaf . Each value is the avera.e;e of twelve tnhibi tor te s t p l ants. It i s thus a.p parent tha t wi th gr owth the total a·notm t of inhibj_tor per leaf doe s actually decrease until :i.t is no long er de tee tab le and onl y e.u..xin cu.rva t u.r es resul t from the ether ext.rac tions. From the :tact that t he amount of inhibi tor decre as es as the leaf grows the CCJnclus i on mi ght be suggested that t he i nhib i t or 1.s related t o the lee.f growth process,. It :t s well to bear i n mind ths,t in vi ew of t he r ec ent wor k on l eaf growth fa c t or s,. ( Bonne1" , Haae;en- Smit , 'l Went , 19.3 9;} le af g:ro1cvth must b e con sidered t he resu lt of b oth vein growth and meso phyll g:row-th. If i nhi bitor were to play a role in le af gr owth ( see Chapt ., VII) it might be ec tl ve in either part t! 26. Chapter IV. A. M r Chemical. Properttes of the In.hib i tor,. Solvents for inhil)i tor e11trac tion. .. c. E r n s t R edemann w o r k i ng w i t h D ;r- . H e. a g e n ... S it beg an an investig ation of the chemical properties of the inhibitor substance (unpubllhshed} .. . From investigations of numerous solv en t s, f01" ex.ample , _.,. benzene ,. methanol .. ethano l:, etc. , he found that a ppre ... ciable amounts of l nhibi tor were extracted by on ly two of them; di ... ethyl-ethel', and ethyl acetate. As noted it was di, ... ethyl-ether that we.s us ed :tn the original ex t raction~ T1:l,1e ethyl acete.te was found to extt'-act nerd" ly twice as mut;h inhibitor as the ether and. also it did not extract as much c h loro phyll. It has recently b een repo;rted by Scb.mauss (ux1published), that 'the etl yl acetate is never- the less a less des i r eab le solvent t han t he ether., The r~a.sons for this a..rQ: l.) 11he sollibili ty of we.ter is greater in eth yl acetate than in ether, 2. } The ethyl :swetate rr,rdr oly-z es' to form a c etic acid, and 3 . ) Tlaat b oth the water that 1:1~.s been taken up by the ethyl ac e tate ~ and even or e so the acetic acid , cause the in.hi b i tor to 1.:n'1derg o a h.y... drolyais as n ill be e xp l ained in the next Jie.rag ra.ph. 27. B. Hydrolys i.s of :l.nhib i tor to yield auxin+ When an e.ctive extr ac t of t he inhib i t or was tested fo:r its stability in h ot hydrochlor ic acid and j_n h ot po .... tassitL~ hydroxi de Hedemann r ound a very surprising and unexpected i-·eac tion ., W:t th t h e alkaline hydrolysis the pos t~ tive curva ture ac ivity was completely los t and re placed by a strong n egative curvature acti vity., Tb.is made it likely that t he substance ce.using th® posi t ive curva ture was a deri ve. ti ve of one of t he auxins. I t also r!lade it necessary to postulate for the struc ture of t he inh:tbit;or a compound., par t of wh ich was this auxin , bu t without its free carb oxyl gr oup as it had been found t hat t he inh i b i• tor contain ed no acidi c or basic groups .. From t he pro ... nounced fat solubility of the i nhi b itor it also seemed like l y t h at t he linkage b etween t he Bu:x:in part of the molecule and the r e-~~aini :ng porti on wa s an e ste:r linkage. c. Iden tifi cation of the auxin .from inhibitor., An attempt was made to ident i fy the nature of t he a u.;;:in produced by the alkaline hy drol ysi s -. This was done by determining its s t eb ili ty upon refluxing in 5% potas.... sium hydroxide and in 5;; hydr och l oric acid, Kog l and Ha ag en .Srntt (1934) hs.ve shown. that under the se conditions of pH s.uxln ... a, is stable to acid but n o t to al kali ; auxin b 28. is not stable to either o.f the reagents; whi le indoleo. ;; ... ac etic acid ( hetero,,auxin ) is stf.tc) le to alkali b ut n ot to Stcld . From this ndiff erentia l destr uction fl t est the evidence was in favor of considering the gro'Wtll promoting fraction to b e indo le ... 3 ... acetie acid., It s hou l d b e no ted, however ,, that this test is not considered :reliable when , there are many impurities preseni- . a s in !t his c ase. i:Po .,a.in further indication as to the ch emical nature of t he inhibitor ., ·nolecular weight determina tions we:re at... tempted . The estimation of mol ecular weights :ts bas ed on the determination. of the diffusion coefficient wh ich is mttde in the fo llowing ma.rmer: Three plain 1½~0 agar pla t® 'S! are stacked one :r,bove the other, a. fourth agar p l ate of the same size , in thi :s c a.s e 1 x 6 x 8 m..11., ,.. and containing the substance being investigated is then placed on top of the s t ack of three . Af ter a given tin1e the distribution of the substance i n a ll four pl ates is deter mined J in t his case i t was m.ea.su:red by the inhi'b i tor ·\~ ·; st but with only a 120 minu t e s instead of the regul ar 150 minutes to tween the ap .. plicati on of the test t loc ks and d eter-mincst .L c:,,-: of the c urvature . ( 'i'his 30 minutes differe nc e wou l d tend to , o.ke a le ss accurate deter.m inat:lon than other wise .} T.10 ,::i.;-i - t r ibut:i.on found in t erms of rel1?ttive concentration :ls then calcu l ated on the basis that t he total a ount of curva- · ture of the f'euz• plates equals 10,000 . From the s e values one may refer to Diffusion 'r~bles by Scheffer and ::~w :-.i 1 ::i 29. ( 1923) and find an 11 x 0 value . This 1 'x" may then b e sub sti - tuted in the followinr;; formulae g iv en by Bruins {1922) ar1d the dl ffusion coefficient thus obtained..,: D = 4 t X in which h is the tb.ickness of the a.car plates in cm. t i s the time in day s that the diffusion~is allowed to continue . x i s the value from the Diffusion ~P ab les mentioned above. Having obta.ined t h e diffusion coefficient D. one cru1 then calculate the mo l ecular wei@ht from. the fol lowing rel ationship as sue;gested. by Oholm (1912): where: I\1 e Vi . is t1"1e mol ecular weight If 11 t emperat ure coefficient ( 1/2?3 ) It " " ~entigrad temper a tur e f! diffu sion coeff icient This meth.o c7. was first used for p l ant hor mone rno lec u lar weight determinations by Went (192'7 ). 30. The date obts.ineo. by I:lr ., Rede.mann raay be caleu le.ted to g ive th.e fo llowing valuen: 1?4, 212,, and 222.. t shou.3'..a be noted that he states t hat all knovm err•ors wou l d t end to ake thes e ve.lue a too larg e. 'l'he values I l ater deter r1ined were ., as predicted, actually lower. See Chapter VII . Prom the mo lecu l ar weight detet-minati.ons it i'v·as con ... cludea. that the auxin der•:tved from the positive curva ture substance cannot be ei~her au:xJ.n a nor b as both of t hese auxins have mo l ecular weigl1ts higher t :1a:n the h1ghe s t value f ound for' positive curvature substance o In su:rrr.ma:ry ,. t h e cher"1.i. c s.l properties of t_ __ e :i:nh i b i tor are: l, 1 That it ca.n be ex t racted f'rom radish coty ledons with either di ethyl e·th er or ethyl acetate . 2. That it contains neither acidic nor basic groups . 3. Th.at i t ee.n b e hydr•oly sed wi tn 5;i potaasim ey .... dro;tide to rield a growth promo t ine;; f nae tion _., 4.,. 'l'hat a differential acid- a l kali destruction t est of this growth promoting fraction indicates that it could be indole acetic acid • 5_ That the molecular i1e1ght deter-minations indicate t ha.t the growth promoting fraction could be neither auxin a nor b, ; and 6• 'I'he. t ind:t e e.tions are 1.1:1.at an ea tcr linkage ex ist s b etween the r.;:rowt h nr ... 0,;:2.a t in,;,; -- ...oart of t h e molecule -.....; and the unknmm part . 31 . Chapter v. A. Growth Inhibitor &nd Growth . G-rowth e ffe c t of' i nhib i tor on Avena coleoptiles,. As mentioned in the introdu.ction, Avena. growth cu:rvatui"es c ~m only b e caused by a r ela. ti·ve difference in g rowth rate on two sides of t he p lant . A ra t e whic h was rela..- tivel y lower on t he treated s ide woul d cau se a posi t ive curvature . This rela ti ve difference of growth r a te coul d b e b r oug h t a b out either by f 1. Jin actual i nhib i tion of the growth on the t r eated s :i.de , or 2 • .By· an increase of t he growth ra te on the untl"eated side . To settle this ques .... tion an d to 1nves tig :;d:;e t h e dir ect e ffe ct of inhib:i tor on e r-owth , growth re.te me a s urErnents were made as f ollows: The upper 15mm , of intact Jwena coleopt:lles we re ma.r l-rnd into 3 mm. zones with India ink . The g rowth of these zones was tb€n f ollowed by rneasnring the increa se :i.n the d i stanc e between the m at ln terv als of an hour and a lml f . r_ro one set of s :i.x. p lants inhibitor wa s a pp lied s -y-:i: ,-i_.... metr i c1:-d ly over t h e tip m:i. J.limeter of the coleoptile in a s mall dro p of l anol in pas t e . ':Po c,n:other set of s i x plant s pur e l anolin paste i:,i lone was applied in a comparable fa s hi on . 'I'he growth increase of these two sets of plants was ~ne a sur ed t:icc ur ate ly by raes.ns of a,.. h or i zonta l microscope which c ou l d 1 e read to a •.0 1 nun. vert i ca l differenc e . '11he pl ants , t b.roughou t the course of the ex per imen t ,were kept 32. in the reg u lar Avena te sting dar k :t"' ooms wl1ere hi;unidi t y e.nd temper ature wer e kept cons te.nt at 85-%, a t1d 84° 8~ They were illmnina tf)d on l -y with t h e lig h t t r ansmitted tD..rough a Cornin g or ru.1g e l .i ght fil t er No , 243 . The res u lts of this det erminati on may be seen i .n fig ..u r es 10 and ll. F'ronl fi g m:>e 11, i t may b$ seen tha t the effect of inhibitor is to dec rease the g ~owth r at e of e ll zones o.f the tr ea ted p hm t s by about 20%. From fi g 1.u •e 10 it is cl e ar l y s een that this dec1.•eased growth r ate i s uniforml y di stributed over the entir e leng th of the p lant . 'l1his e.:xpe rim.ent was repe e.ted u s ing i' pur if ied " or auxin :fr e e inhibi tcH:· in l eno lin . f'i c s.tton method}. ( See Chapter VI for puri - Thi s test clif f'er ed from. t h e fo r mer only ln t h ~Jt here t h e pl ants were older at the beg :i.n :ni ng of bhe exper iraen t being 3 0 mm . tall as c ompared t o 25 mm .. in t he oth0r ease . T'n e res ul t s of thi s e4'Cperiment are s een in fi gures 12 and 13. While they are essenti ally the same as t ho s e obt ai n ed with t he un purified inhi bitor they show a gr e:.;: ter runount of inhibi tion a s in thi s instano e the grow th is comp letely sto p ped in .. six hours,. F'~""o0-11.re . . 12. s h ows that for t he f iJ•st two hours af ter the application of' the pu:rif':led inhibitor t he gr owth ra te of all of the zones i s li ke t hat found for t he unpurifi e d inhib i tor but .f;ror11 that time cm it deer eases more ra pidly. Corn.paring fi gure 13 with fi g ure 11 one sees t ha t there i s roug hly a 20%' g rei1te::r growth inhibition effe c t of the pur•i f ied inb. i bi tor 5. 4. 3. 2. Zone 1. : I : I : 10 . 3mm • to -=::::::=====::::::=====:ti =====::t~======li======!:::! ==:].A. -ti,esoco ,l-_ y l • 4m□ . 6mm. iJ.2nm;. · 14mm. '16mrr, Zones of Coleoptile. Fig . 10. Growth Distribution of the Aven~ Coleoptile when treated with Inhibitor. r:n k ;j 0 .c: ,jN r-1 ---...... .G .µ Ql) s::: (I) rl s::: ·rl (I) (/J I • .-----·----.---·------·- -------·-========:.:..~Q.?,n t r o 1 bi t or 1 - - - - - - - - - - - - - - - - - - - - - - - - - - _ _ _ _ ; ; ; ; . : :In.hi ,; --~- --- ·- - - ____ :=:::::::::::::======:--=-=.::.::.:::.:::::::::-::::~=::Q_~o.n t ro 1 i - - - = - - = - - - - - - - - - - - - - - - - - - - - - - - - - h i b i t or :'.'I (!) H 0 s::: H ·-----:: ___________ : _ _ :-----=====:::::::-::::::C~c,>_n t r o 1 ~.L------------------------+-¥1-.hi bitor TIME (hours) Fig . 11. Growth r at e a nd its distributi 0n over the Avena coleoptile during 12 hours when tre at ed with inhibitor. ,..-... co H S:::_q •.-1 30 tO (l) .......... co ti ..c: •J) 5. 20 4. 3. +' H bO 0~ s::: Q) Hr-i 10 -~ I I <:: I ll:nmi . Fig. 12. 1 • knm,. ~2mr!!. 11.03 rnm. to !16nm~. metocotyl. i '14mm.. Coleoptile zones measured. Growth distribution of the Avena coleoptile during six hours when treated with 'purified inhibitor. ·--------- ---- 2nm,. ·---.:...____ , ~_ntrol - - - - -- Inhibitor ·---.....___ ·----------....;._-~QJ~nt rol ---..._ ____ .Inhibitor s::: ·----· •.-1 ·-Inhibitno~r:-------~ - - -· Control Q) +> ra 2mm. ..c: ,µ 2 F-1 s:0 •--------=-: F-1 C) 2lll!!l. Fig. 13. Time in hours. Growth rate distribution over the A~ e na coleo ptile when treated with purified inhibitor. 33. over its eontrols than wi th the un puri.f'ied inhib i t or ,., This is apparentl;y due t o t h e f ac t tha t t h e unpu rified in-h i bi tor contained a ux.in tog e t h er with oth En"' i mpuri ties •. B. Obs erved positive curv a ture ex pl ained. on a bas i s of mes. s ured growth inhibi tion., Since t hese res ults ind ic ated t hB t the inh i b j_tor acted on the Avena coleoptile solely b y causing a trrowth t;, Z, inhibition it was :i.nteresting t o see i f this conclus i on c oul d be subs tantiat;ed by using it to pre dict the amoun t of po sitive curvature obtained by the uni l a tera l applicati on of t b.e inhib i tor . This c oul d be done by c a lc u l atin g the amount of positive cur va t ure to b e expec ted if the de cr e a s e d w> rowth r ate deter ir.tined ab ove were to act by caus• ing the same growth rate d ecre a se bi1 t .,, only on one side of E>.n Avena coleoptile . To ans wer• this po i n t the s ame c oncentr a t ion of unpuri ... f ied i nhib i tor in l D.nolin , whose ef'f ee t in decre as ing the growth rate is given in f i gure 11, was applied unil a t er~ a lly over a 5 _,rm. , strip , 15. 6 mn1. b elow the t ip of 25 rmn . Aven a c oleopti l es o This mean t that t he inh:Lbitor was ap ... plied where zone 1., woul d be on t he plants i e t>.s u r ed fo r t he growth r &- te re s ponse . For this re12w on i t wa s nec es - sary to us e the growt h r a t e of zone 1 in calc u l ating t he amount of' positive cur va ture t o b e expected from s ueh an i nh:tb i t i on . r his d i ffe rence in grm-vth r at e was found t o 34. be ,. 5 mm. for the firs t two hours. By substituting in t he for mul£?.e: where: dis the dis.meter of the plants i s the a.ngle of the cu1"v 0 tt'J.r•e lx is the gr owth r ate of the convex side J· V. is the growth ra te of t he concccv e s ide a it is possib le to ca::.cul ate t he expec t ed curva ture res ul t ing from such g rowth ra t e di f f erence on t vm stde::-, of e. growing org ru,1 ,. The averag e diameter of the plants which were unilater•a lly treated Vlas i' otmd to b e 1 . 4 rmn . by direct: meas urement . 'lli.e growth rat-e cUfference a.s ment lonea ., vrns . 5 mm. for the first two hours , hence by s ub sti t uting in t he at:ove e qua ... t:i.on. a11.d s o lving for the ang le of c urva t ure, t hat it i s suppos ed to equal 20 . 4 0 11 a " , we find positive curvature . By ac tual measur ement of the pl ants trented unils terally with the same concentration of inhibitor i n lan ol in ., after two hours the posi ti ve curvat ure Wfis f oun d to b e 23 . 8 0 From the above dtetta. , then , :i.t is a pparent that the posi • tive curv atur es re sul ting from the rela tiv e gr owth i nhibi .. t.ion eff.e ct of the i nhib itor Rre ac tu ally the res u lt of a g1~owth inhibi t ion on the trea t ed sid e . It is also seen that the growth rs.te .me:a s urements f r om plants treated with inhi - bitor can explaln the positive curv atures obtained b y the 35. unile.ter•al applieation of i nhib itor . c. Grov.;th eff ec t of .inhib itor on tissue sections oi' Jwena and radish. I t was in t er e s ting to deter mine the e f f ec t of inhibi t or on the gr owth of 4 .• 0 mr.1 0 sections cut from Avena. coleo ... ptile s . It is lm .,wn t1.8t when suc h sect t ons are put i n incre ases over wnt er coµtrols,(Phytc- auxin s.o l ut:i. ona their gr owbh norrpone s, 1~3? ) It is a lso known that the ar.<1ount of t h.ts grow t h is pro- portiona l with in limits to t h e concentra ti on of t he 8.ux;ln s olution., Ii'or these reasons a c oncen tra tion seri e B of an active inhibitor ex t ract made . The c otyledon ex t ract wa.s taken up in 4 cc . of wo.ter and successive t enfold di l ue tions were made down to 10 of the hi ghest c on cen.tr e.ti on . Two cc . of e a ch of t hese concentr ations wes t hen put in a ~-'.yrac u se dish and thirty 4 ~0 mm,. sections c ut f rom the up ... per half of Avena co l eopti les were added . The s e di she s were kept in t he dark r oom until the ti me for t h e grm1th me a s urements 24 hours l e.ter . A , this time the y were t aken into the l i c;ht an d rneesured a (: ,:;ur atel-y to a .1 r11.vn . by means of an oc u lsr :ni c:rmneter. ment are seen in f i gur e 14 . The res ul t s of this experi - Here it is noted t hat t he high ... e st concentration of inh:Lb itor e e.u sed a s hrinkag e in the ori g ina l siz.e of the eell . Thi s wa s due to t he fa c t that t h e soluti on wns tox i c a s the sections tier e flac c :i..d. .. 'rh e •.. _-=-~~:-:-:-:-;;;,- ---~--- ~-~-~ ~~--~- ----:z.~ =-~:_con t r ol ~ -- . Innibitor 1 concentratiun Fig. 14. Growth of 4 furn. sections of ~ven~ cule optile in inhibitor .solutions for 24 r, -::)UrB. ~ () -~ .p 0 (j) U) -5 l -4 Inhibitor con6entration f ig. 15. r.:: 0 ·r-i GroTTth of 4 mm light grown sections of Radish hypocotyl in inhibitor solution for 24 hourso - Control Inhibitor .µ 0 (I) U) 1 -5 Inhibitor concentr~ti~n Fig. 16. 1. Growth of 4 ri;n,. d "irk gpo,··n sec : ion of Ri..i.dish hypocotyl in irwibi to :. solu.ti.on for 24 hours 0 36. other c oncentra ti ons inhibited t he growth of t he secti ons wh en com.pGi.red to t he r r owth of the wa ter controls except at t h e very loiivest concentration.. In that case t h e growth of t he sections in t h e inhibi t or and :l.n the w1:1ter were the same~ There was no relation fo und bet wc \7.m t he eoncent;r,a ... t i on of lnhibi tor and the amount of inh.ib5. tion., rl'1'.n.i s experi• men.t has been repee.ted t w:l. ce with essentially the sa.m.e re ... s u.1ts., tions f ro:.;1 radioh hypocotyls a t t he same inhj_b i tor c oncen... trfitlons ,_ s a.bove . 11.' he radish p lan.ts bed been g rmvn in tlle gr eenhouse in the usual manner and wer e 5 days ol d at the time of the ex tieriment .. Ano the r se t of radish pl an t s we.SJ c;rown wt1ieh wer e et5- o l e.ted by keepine; them in the dark rooms u sed for Avena te s tir1g ~ light 1;:u-1 d 'J'.11.e res u lts for b oth the dark '-· .c.: rown recdis.h sec t :i.ons ·wor e $imilar t o tho se ob te.ined with the Avena coleoptile .!H~ot:tons . Tr.ds dat a t s presented in .f i gures 1 5 .,. a nd 16 .- In conclu e i on it may be said that the inh i b it or acts on t he gr owth of the Avena co1eoptile to c aus e posi tiv e curvHtn.res by s.ctually c ausing a growth i nhi b iti on . The Ennount of inhibi tio:n ;i s ~m.fficient to ex:)la i n the po sitive curv ature s obtained from. the unilateral application o:f inhibitor at th.e middle of the coleoptile . Inhib itor likewis e inhibits t::~e g rowth of isol &ted :;,ec tion.s. of Aven:;,, coleopt i les and .r adish hypocotyls 0:rnept 37. a t the lowest; di l ut .L ons 1?1here_, in the cas e of the radish , it cau sed a gr owth promo t ion. There wr,s no linear propor- tionality betwe en the con,~ entrv.tto!'l of the lnhibi tor a nd t he amount of s ect i on e r owth i nhibition . 38. Chapter Vl. A, Inhibitor ~Prans port Th't'oug h Avena and He.dish Sec tions . Non- po l ar mov ement of inhi bitor in Av ena co leopti les and radish hypoc otyl s . 'rh e inh:Lbi tor s ubs tanc; e h as b e en s hown to b e of n eu- t ral chemical c haracter, i.e. it contains neither acidic nor ' a sic groups , hence L 1 r e ,, ard to t h e polari t.y of it s t ra;.."l~pd>rt through plant tissues on the ba sis of' V!e.n t I s (1932 ) potential gradien t theory fo r polari ty of plan ts, one wou l d ex pect i t to b e trensported ~rnropetn lly as well as basipetally. According to this theory only certain or- g n:tc "Cids for exampl e aux i n ., s.h ould move basipetall y whi le b ases should move onl y acropetally. A substance of neti tral character s h ould b e tran s po!'ted e qua lly well i n e i t h er di -- rec tmon,. The f ollowing experi ments s howed thi s t o oe the c as e with inhi b itor substance. Two pl a tes of b lank 1½% ag ~r, lx6x8 mm. were placed e ttch on a seps.rate micro scope slide. On t op of' t he p l ates we re pl aced on e nd .,. twe lve 4 ~2 mra . long s ections of Avena c oleo ptt l es . On on e of t h e pl ates t he se were left in the normal posit ion (i.e. in the s ame position with res pect t o g r a.vi ty as in tl.1.e p l ant ) whi le on the other p l at e they were inverted~ On t op of b o t h sets of sections another ag ar p l at e was pl acec1 simi l a r t o the lower one b ut c ontaini ne; 39 . the inhibitor ('' donox> pls.te 1t ). Both slides were then put in a Petr i dish in which there was a s mall pi ece of :moist ... ened fi lter p aper. The who le procedure W8.s car>ried out in the r egul ar .Avena. dru•k ro om •. After two hours the pla tes were cut into 1 x 2 x 2 nun . b loc k s and analyzed f or :Lnhibi tor . rrne reeul ts wer e as f ollows: Coleontile Position ( Upper plate, inhibi tor donor) CuJ:vatu:r>e , Avg . 9-12 test plants With Stand. Error .. Normal~ lower pla t e 4$4 ± .5 Normal , upper plate 24 . 0 ± 1 .. 7 Inverted, lower pl ate Inverted, upper pl ate From t:hese resu l ts it is concluded that with the concentr ati on t1sed in this e x periment ., there is n o pol a:r-i ty of :nhibitor transport i n the AvenH c o1eopti l e . Other ex ... periments to be discussed s oon verify thi s conclusion . It s h ould be mentioned that t he poler t:re.n sport of auxin is un .. affec ted over a very l ar ge concentration rang e., o .cording ly ive may expect the same to be the c as e with the non- polar t r ansport of inhibiter. The polarity of inhibitor t r ansport was next i nvesti ... g ate d in 4 .. 2 mm~ s ections of radis h hypocotyls , The ex peri- ment was performed in t:he same :manner as described abov e fo r the Jwena colec.1pti le s exce pt t ha t radi s h hy pocotyl 40. sections were used e Duplica.te d e terminations we .re ·1_ade ., The res u lts are 0 i ven below: Po si ti on of Hypocotyl {Upper pla te , inhibitor donor) Curva tv..re,. (Avg • of 12 te st plants . ) I II 0 . o Normal , lower pl at e 5.,0 r,. or ma 1. , upper plate 13 .. 6° 11 .,6 ° Inverted, lower p l ate 3.6 ° 3.8 ° Inverted , upper plate 14 . 1 ° a~.,O o 3.1 Thes e hypocotyls were f r om 6 day ol cl plants that had been grown in the g r een house under the re'-;u l ar c ul ture condi tions. Before pla.c:i.ng the hypocoty1 sections on the ag ar plates they were allowed to stand on wet .fi l ter p8.per for 30 :ninu t .es to tend. to empty them of auxin and to 1"'emove d e- s truc ti ve en zymes frorn t he cut surfa..c es. f rom the. above dat,.' It may- be seen t ha t radi sh hypocotyls like J\vena. co- leo pti l 0s hed no transport polarity for inhibitor . In one exper iment ., performed under condi t :t ons wl ·.ch were presumab l y the same as those above, auxin was found to b e pres e nt in the receptor ag ar p lates of bo th the normal and inver ted sections. The actual d ata f'or these surprising results , was : Hypocotyl Position Ct;trva t u1"e, ( Avg .. of 1 2 t est ( Upper pl ate inhibitor pla.nts with itan~1. Error) donor) Normal, lower p l ate 41. 0 No1"me.l, u ppe::r pl ate + 15.0 ± Inverted, lower plate • 8 "8 Inverted, upper plate + 11.8 0 1. 1 :t: 1.-:0 .±. 1 .3 0 P, ttempts to repeat this experiment showed only i n hibi - t or passing through the normal and. inverted sections .. B. Chronoloc ical record of ·~ov ement of inhibitor thr ough sections- of .Avena co1.eo ptiles and radish hypocoty ls ., Since it was not ,,o : sible by the above meth od to get a chrono lor.;ical determina t ion of t he eff ect of passing in~ hibitor through radis h hypocotyls and in view of t he sur prising results obt ained in one instance., it was f ound d@sirab le to m.a1~ such a determ nation" I 1h is v,as done by mount ... ing a 4 .,.f .rnm. sec tion of radish hypoeotyl on t h e shoulder of a d.ecar i tated Avena coleopt:tle . A very small s pot of Vase- line was applied to the prira.ary leafl to held the sec tio.n upright on t 1e t op of' the coleop ti l e . On t op of the radish sec tion was then placed an agar block ( 1 x 2 x 2 .mm.) con ... taining the inhibitor. A silver w5. re wa s i.nserted into the primary le af so the. t t. 1e who le set-up could b e placed in fr on t of the kymogra.pb. drum. as described in Ch.apter II .. By this method e.:ny growt h promoting or inhibiting sub-. stances diffusing from the section woul d pass on into the 42. Avena coleoptile and caus e a growth reaction Whi ch would then be re cor ded.. Four a t tempts were made with thl s tecbni que before consistent data were obtai ned . The reason f or not obtaining good results was probably due to lac k of skill With the method,. It Wt=ts d ec ided th""t before investig a ting tb.e effects of inh:1.bi tor t r ans port throug h i nv erse s ecti ons of Avena and r ad ish with this method-, the transport in the nor ... rnal d irection .S'houlcl be well understood. 'l'oward this end 2,.1 rmu . Avena coleoptile sec tion.s were c ut f rom ne ar the tip and near the b9.s e., I1l0se ' vere then te sted , by the kymogr aph t echnique described above, f or the effect of inhi bitor transport. A sim.i l a:r. s et of 2.1 rll.l'!l . radish pypoco tyl sec- tions were also inves tiLe, ted i n t he same way , at the same timth The res ults of these experiments a.re s hown in f i gure 1 '7 .. Here it i s clear ly seen th.at in b oth instances the ac tion of the inhibitor ex t r a ct was to cau se at fil•st a strong negative curva ture which t hen c urved po sitively. Only in the case of t he sec tions cut from ·t he base of the r adi s h hypoco t yl did t he cmwatur e s actually become posi tiv e, howeve:r. When the inhibitor used in t hi s experiment was t ested directly on the coleoptile i t g ave a posi t ive curvature of 6 •3 degree s in a 1:4 dilution.. 'I'his indicate s tha t there was sufficient :i.nhibi tor in the top b l ocks on the secti ons to caus e 25 ,.2 deg r e es posi tive curvatu1•0 . Thes e res ul t s are, however, open to c:r:tti cism as there Negative Curvature. 5 n:iru . 2 mm. ll.lILl. mm. sect. Ililii • tip F ig. J.. '/ . sect. rleac t ion r~ t e t o inhibitor transported through Avena coleoptile and Ra.dish hypocotyl sectionso (normal position, 2.1 mm. long.) 43. raa;y have been ~uxin in the crude extract which cou}d be transported through the sections and c~use the observed nee;av ive curvatur e . 'i10 overcome t he objection the follow,.. ing method of inhibitm:.. purification was devised . c. .iuxin con tamination of inhibitor ex tract ~1.nd its l\emov al by the nintreJ:>se Transport" puri fi c ation method .• Since it had b een s '' O\"f.i1 b y Redemann thet any of the usual c hemical treat,,1ents used to remove auxin from the extract :r.e sul ted , not in the removal , but in the hydro ... l y-sis of i nhibitor to yi eld even more e.u...~in , c he r, ical tre17-t• ments were not considered . It v.ras also n ece r, s a.ry to de-• cide how to deter mine when all of the auxin was r emoved . I t was assumed that if no initial :nee e.ttve curvature preceded th_, posit i ve curvature then there was no auxin pr esen t , o r t he. t if it weTe present it was be low t h e sensi ... ti vity Jf t he plants and so for the pur pose of subsequently detecti ng auxin effects it could oe regar ded as neg ligible • .. inc e it was :impossible to purify the :tnhibi tor by knovm c hemical means wl t hou t rc-i.nning the risk of hydrolysing it into auxin , a physio l ogical purification method was de ... veloped .. It is well established t h& t Aven a cole opt:. . les wil l onl y transport a u,.· d:n bas i petally ., Onl y when t l1e auxin 44. concentrations are extremely h i gh ., -- far hi g h er the:.11 those deal t with here-•, only then could the auxin pas s through Avena sections acropetally . Inh5.bi tor substan ce on the oth er hand a.s we have alres.dy seen can b e tran s ported in both directions of the Avena coleoptile -. ity of transport ~ It has no polar ... By using tb.ese t wo facts : 1. Au.x in t r ansported only toward t he bas e of the coleopti le , and : 2. Inhib i t or t ransported toward the tip or ba s e., it was thought tha t i t i.n:Lght be pos sible to separate the inhi - b itor f rom contaminating auxin by passing it throue;h Avena co1eoptile sections fron1 bs.s e to tip and thus keep t he aux.in from passing :nhrough . following manner: Tb.is was t ried in the 'l1wenty 4 . 2 mm. sections of Avena coleop- t iles were inverted. on a b lank ag ar p l ate e.s EHU"'1ier de.scribe-cl . On top of these inve;c>ted sections was then p laced an other ag ar plate wh ich conta:tned both inhib itor and any contnminating auxin .from the crude extr ac t ion . This 5et up was allowed t o rem.a.in thi s way 1-·or ten hour· s • At tlle end of thi s time the i nhibitor in the bo t tom ag ar pl a te was te sted on the kym.ogra.ph, i n two con cEmtrat"' ons, l and 1. 4 • As can be seen f r om figure 18 . (Each. point a.ver a 6 e of 11 test pla..?1ts on fu ll str en~rth eo:ncentrat:'!-on curve., aver-ar e of 9 plants on ¼ c oncentra tion curve . ) no nega t ive curvatures were present in this way . 2. ) when the i nhi b itor was puri fi ed {Compare with unpurifled inhibitor , figure r }:\...is experiment was repeat ed tx1re0 times vd t h the 1 Fig,o 12 . Reaction rate to purified inhibitor. Two Concentrations tested, 1 and ¼o Twice Deca~itated Avena test plants used. Negative C rvature. rum. 6 lll:U .• lilL. 2. mm. 6 l'lim. mrn. 12 mm. 15 mm. Positive Curva.tureo 3. Hours 45. same :res u.1 ts in every ca.se . It was concluded t h at eon.-. taminating aux.in was r emoved .. In s ubsequen t work when r eference is made to purified inhibitor it wi ll b e under .... stood tha t it was pur;i.fied by thi s ninverse Transport" method., D. Action of .Ave na t es t plants t o i nhib .i to:r contarni - nated with auxin~ It was ne1ct interesting t o s ee if t he action of the crude :tnhi1'.) i t or ex tr(a.ct, ....... a n eg a tive curve.tu.re followed by a positive one , coul d be duplic.a ted by using purified inhi -. bitor and mix ing with it indo le ace tic acid ; a growth pro:noti n g s ub s t ance. the kymograph: This exper iment was done b y testing on 1, purified inhibitor alone; 2. 4/5 puri• fled inhib itor pl u s 1/5 of' en ind.ole a.ce t i c acid solution of a. concentration of 45 g a.IT1..."lla per l iter, and ; 3. 1/5 inhibi t or pl us 4/5 of t he :indole aceti c ac id solu tion .. The. results are seen in figure 19. Eac h point is _t he a11rer- a.g e of 10~12, t wice decapitated. plants. It is clear that t he same type of curvature :ts f ound as that ob tained wi t h the c rude inhibitor extract . It may then b e conc l ude d t h at the neg ntiv e curv e.tu.res prec-e ding _ the positive cur- va tures and res ulting f r om the app :i 1eation of t he crude unpurif:l.ed inhibitor extract , are d.lle to aux i n whi c h was e.xtre.cted f r om t h e cotyl edons tog e ther wi th the inh j_b i tor. l. t .. ,.. ...J L..,•_ ... . . i .. ..... .... .,_J 46. It should be poin ted ou t that when lt was found t hat 1.nh:t ... bitor was presen t in t he vegetative buds and young leaves as ¥ell. as in the cotyledons , :rnaFy of the crude ext r a ctt3 were rnade by using the who le radi sh plant except for the :roots. As auxin i s pr es ent i n t he hypocotyl this proc eed;,,- ure a llowed a ,uple oppo:rtu:n ity t o e:xtPe.c t auxin a long with inhibitor .. Moreover ., :1. t has been s ho-vm by Van Over b ee k ( 1933) i th.at it is even po ssib l e to ob tG..:tn auxin f rom the radi sh co ty ledons the mse lves try the diffus ion method of' auxin extraction. . According to this me thod the cotyledons are cut f rom the p l ant ne ar the hypocotyl e.nd allowed to • diffus e out ru1.y substance s into 1 x. 2 x 2 inm . 1 ½ ag ar b loc ks pl aced on the cut end., These ag a:r b loc ks c ause ver y s t rorig auxin curvatur es ln the Avena test~ r:!:his indicates that auxin i s comi n g f1" om the cotyl edons • 'fhe point to be made i s t hat th.is auxin is prob ab l y present in ·the crude extract but that in the stand~rd inhi bitor :best its presence would no t b e noticed bec aus e of t h e obsc uring ef'fec t of the 1ar... ger amounts oi.' inhibi tor; howevew a kymogr aph te st , would :r e cord the pre s ence of auxin. By direc t observ ation of t he test pl ants it is possible to s ee the negative cur va. ... tures ree c>rded by t he kymog raph11 'J:his is _parti c ule.rly pos sib le in cas e s \.Vhere the a moun.t of inhibitor is not EH!.., pec :Lally h i gh . Under such conditi ons i f a measurement wer e taken of the amount of :ne ga.ti ve c urv at;ure it wou l d s erve to anner som..e of the s ame ques tions th~tt are an swered 47. b y the kymograph testt i.e .. namely, now much auxin and how mu ch inhi b itor i1.re:, pr .-;,s-en t :Lr1 t ho e x.t1.•act. 'lt'llhen inri.J.bi tor extre.c -t eon tains auxin in t h e proper rur1ount it is pos.sibl® to obtain t es t pl ants which s how S s h aped curvatures after a.1'1 hour and a h e.li' • These c urva• tu.res may b e interpreted as being ca.used by the auxin mani.,. f esting itself first to give a growth promoting effect hence a negative cn.rvature Which wi ll move downward: into the l ower zone s of the coleoptlle w:M. le the inhibitor acts to g ive a positive eu1"vature in the upper zones .. Examples of tbi s type of curvature are seen in fi gure 20 ,. At t h e end of 2-l hours when tltere i s a l e.r€Be conee:n tration o.f inhibi to!', the S sh aped eu.rviiture will b e en ti:rely re plaeed by a com.. plete ly positive eurvatu:re. E-. Curvature rates of Avena coleoptiles t o di lu• t:Lons of i:nhlbi tor .. The gre Hter t he d.iluti on of the inhib itor the less ia the ne t amoun t of positive curvature and also t he gr eater is the negative c ur vta t ure if' t her e i s au xin present. This is seen i n fi gure 21 . Ni ne to twelve test plant s were aver- ag ed for each point ., In thi s e.x.peri men t , a di lu ti on series (1, 1/ 2, 1/ 16, & 1/ 32) of crude inhibitor extrs.ct was tested directly on the coleoptile by the standard kymograph t echnique . From t he res u l ts i t is clearly rrn en th"' t after •L I - ~~ ; ', ;,-- -· ~ S 11 cu1·vaturea an hour and a half arter application of inhibitor containing auxi::. PJ ,::, ,--- - • i•:i ,9 decapitated. 11 Positive curvature of ~vena coleoptiles 22 hours after unilatera l application of inhibitor in lanolin at the mi idle of t ne , coleoptile. Note a lso -he geotropio t ip response. Fig. 21. Curvature rate of once decapitated Avena test plants to different concentrations of unpurified inhibitor . Negative Curvature . I : 9 mm. 6 mn.. 3 3 mm . 6 mr:i . 1/32 mm. 12 IDI :, o 15 mm. 18 lliiL o Positive C irvature. 1.0 48. an hour and a h&lf the pl ants with the 1/32 inhibitor s howed a negative curvature of about ... 5 degr ee s whi le the plants with t he inhib i tor full {,treng th showed a posi ... t i ve curv ature of about 6 degrees.,. .Af t.e:r t he n e:.'!t 2½ hours, h owever, the l/32 conc entration plants had become posi tively curved to a.bout 7 degrees and the full s t rength p lan t s t o "bout 18 degrees.. rl1his data thus agrees with the e.?tplana tion .f' or t!1e oec ure nce of S s bE.ped cur atur e s a t low .coneel'ltr.ations of i nhi b itor c ori ttilini ng ~u.xin • . .' ince the 2.,.1 mm .. Avena a eo ti ons t es ted on the kyr.ao- graph fo r i:nhlbttox• rea c ti on o.iffused out etucin to a large exten t, as w&.s seen l n i), p:-eevi ous e xperimen t,. i t was t hought wor thwhi le to investig ate the effect of' t r e ating see tions longer th~ 2~ l :i:m-n, with inhibitor. This was done in the same manner as deaerib e d for the 2.,l mm .. sect i ons ex cept that the sections (of different lengths) were all cut from t he u pper half of the Avena coleoptile. 'The :re- s u l ts ., of this ex.perimen t are seen in fi gure 22., ( Blank controls- for all secti ons were run but a.re not g iven on thi s graph as they would not affect any of t h e conclusi ons drawn.) It i s to be noted that the longer t he secti on t he 1es s a.u.."tin it transmits ; ( indicated b y t he m:m., of negative c urva ture given in paren t hesis), This c an be understood ii' one assumes with Van der Wey (1933) and Van Overbeek (1939} t l:uit :tt is possible for the p lant to tl se or destroy t he auxin as it tends to pass through tb.e pl ant sect:ton . Fig. 22 . Reaction rate of oncedec ap i tat ed Avena coleoptiles to iilb1bitor. transported through coleoptile sections in the norrud. l position. Each point is the average of seven t ~st plants. Negative curvat ure. 3 rnm. sec t ion ( 48 ) 3 sec t i on mr.o.. 9 filll, • No section,inhibitor Positive applied directly on curvature. the ooleoptile. run,. sec t ion (2) 49. Thus a short section would use less auxir.. than a long er one, while the longest s ection wou l d be capable of using the moat auxin and hence would gL e a :rec ord of transmitting the least., This assumption would also explain the differenee in the reaction to unpur:tfied :i.nhibi t or of the Avena and radish sect i ons cut from the rapidl y gr owing zones, apical sections, and from the s low gr owing zones, (basal secti ons). The rapidly growing zones would be caps.- ole of using more of the auxin and henee would tra.n smi t less t han the basal ! ?.Ones,., This was actually found to b e the case as was seen in fi gure l9. The e f fect of t r~msport of unpurified 1nhibi tor throu.~,h Avena secti ons in the nor mal , basi peta.lov.s di rection seemed now well es t ablished. The pr-e;sen:ce of the auxin curvatures could. be as.c r•i 1 d to a uxin in the er•ude extrac t ,. Likewi se coul d the pre.sen ~e of a ne.g ative cu;r>vature pr ec ed ing the posi tive curva tu e , when ';he agar test block was applied di- rectly , be ascribed to c ontaminating auxin for when the a.u.;,"tin W"s removed by t h e Inver se Tr ans port purifi cation tl:1.ese negative eurve.tt.1.r•es wer e no longer present . The1"e we:r'e still two point$ unset t led, however, concerning the transport of purified inhibitor . The fi r st point was to determine the t r anspo·r t of purified inhibitor in the nor- mal direction. The sec ond point was to conclusively answer the questio:n as to whether o:r. not auxin diffueed out of the inverted sections a.s indi cated by one exceptional radi sh 50. polarity test. F. Transport of purif ied i nhibitor in Avena coleopti le secti ons~ Thes e questions were answered with res.pect to the Avena coleopti l e sections by t he f ollowing two ex . . riments . In the f'i rst experimen t both, m. 1. mm. normal poai ti on sec - ti o"1s and 4,2 nnu. inverted s ections were tes ted for tr$ils po rt of pu.ri.fi.ed i nhib itor by t h e kymo-g raph technique. The resul ts are s een ln figur e 23 where each potnt is the averag e of 9 or 10 plants . It is noted that in both in,. s tances t here is ran aux.in curvature present. Since auxin free inhibitor was used~ this neg at:i.ve curvature .mu st he.ve been the result of the tran sport process~ It is e asy to be lieve that in the transport t h.rough t he secti ons in the norm.al pos ition s ome of the inhi bito:P e oul d have been hyd.ro• l ysed to auxin which then would diffnse on out , bu t in the c ase o: t he inverted section i t is difficult to see how any eu.:-cin c ou.ld dif:fus e ou t, as it woU.l d be going ag a i nst the n :1-ll- estab lish e d po l a rity off auxin trans port. To e.1t• plain this n eg a ti ve cur vature one may as sume t hat inhibitor wa s t ransported throug h t he inv•erted section as inhi bitor., ( wh i c h we h ave seen :ls poss ib le), and tha.t actu a lly no auxin we.s t rans pol'Jt@d aga inst the polarity,. bu t t hat at t he bottom cut surface of t h e sec ti on and t he top out Negative curvature. 3 mm. Ho mm. 6 mu1 normal section 0 4o2 rum. Positive curvature. Fig. 23. section Reaction rate of twice decapitated Avena coleopti~s to purified inhibitor transported through 2ol mm. coleoptile sections in normal position,and 4.2 nim. sections in the inverted position. 51. surface of the eoleoptile there was a hydro l ysia of inhibi• tor to auxin . Thi s might have corae about by t h e presence of hydrolytic enzyme s as it h as been s hown by Van Over b eek ( 1936 ) that at c ut surfaces of Avena c o leopt i le s t':1ere are enzyme s libers.ted , (He fo1._md the pre~enoe of peroxidase). To verify this e2tperi en t it wa.s r 0pea.ted in the .follow... Avena c oleo:pt i le sec t i ans were Q"u. t from ing manner: 4 . 2 tr..m.. the uppe:t> half of coleopti les and w·e re mounted in the :tn... verted position o.n test plants in the manner previously described.. To one set of twelve such plan ts was applied an u.npurlfied in..riibi tor made from the e.xtrac tion of the coty ledons. alone. To s. s imilat- set of plants blank l ½~t ag ar was applied. To a thir d set of six plants \vi th i n ... verted 4 ~2 mm. s ections, ag $.r con tai n;tn.g an indo le acetic acid solution of 500 g a ml:,""'18. per lite r was ap plied, while ton fourth set ,also consisting of si.x plants,the same con ... centra.tion of indole acetic e.cid was a ppl:i.ed dil.. ectly to the .&wen s coleoptile with.out any interve ning inverted co• leoptile section. Finally in.hi bi tor of the same concen tra- tion as that used above and ·b lank aga.r II were each tested direct l y on sets of six p lants, These were all run on the kymograph mac hine a t the s:ac'!le time so t hat any f actors of varying se:a.sitivi ty of the test plants to inhibitor or a uxin were r u led ou t,. The resu l ts are seen in figu re 24. Prom this f i gu.re the following points are evident: 1. That in agreement vri th Gorter' s ( 1927) statement blank .ag ar can Negative curvature. Indole ace t ic acid applied directly on the coleoptile. No section intervening. mm. Fig. 24. Reaction rate of twice decaP,:... itated Avena coleoptiles to indole acetic acid. and inhibitor -transported through inverted 4.2 mm. coleoptile sections. Hours. 3. ·----. I,I¥iole. ~ q i d l 500 g anim3. T I: J rrin i • 2 Il!lll o Positive curvature. Inhibitor a pplied directly on the oleoptile,no sec t ion intervening. Inhibitor 52. c a.use posi tive cu rv atures after t wo hours and e hal f ,; is no t r ansport of pure aux.in alone through the invert ed s ection ; 3 ., Th a t t he in:b..ibi tor extract h ad li ttle a ux .i n in it its elf, •-ah ow.n by t he test of inhi b i tor directly on t he eoleop ti le.; 4 •. Tha t in agreemen t With the fo r mer exper'.lm.ent , inhibitor tre.nspo.; rted thr ough the inver ted Avena s e-ct •: ons c aused a negative c urvature to precede th rhn t this positive curvs,ture oceurred 1 at t he same rate as the posi tive curvature resulting from the ,d.i:rec t a ppli ca ti on of th~ i nhibitor thus i ndic ating thiilt 1~he t r ansported inhibitor still a c t ed the t:H1me as t he non-. transported i nhib i to:r .• G. Transport r ate of purif'ied inhibitor in Avena eoleoptiles. From thes e t wo exper iments it is po.s.s :Lble to deter mine t he r ate of transport of inhibitor thr ough inverted s ection of Av ena coleoptile . The beg innine of t he e umotor ic phase ( se e Went and WLite, 1939 ) is taken as t h e moment of the be ... gin:] ing of the neg ati ve curva tur e preceding c ur vG. t ure, T}lis is based on th t h e posit ive, assumption t hat t he fi rst i nhibitor t o pass thr ough t he s ection become s hydr olysed , as expl ained,. at the c u t s urfaces and hence continues on into the co leo pti le as auxin alone, 'l 'he s ubsequent posi- t ive curvature of t h€l .&wens cio leopti le may b e exple.ined as 53. resulting from more inhi bitor being trans ported t hrough t he section than can b e che.n g ed into a uxin at th<:i cut sur• faces " It has been found. by Wen t and White (1939),. s nd can also be determined f r om fi gure 1., t h Gt the time required for t he b eginning of neg ative curvatures in .Avena plants in 1?c s ponse to indole ... acetic acid is 3? minutes . According ly the 37 minutes re quired for t he initiation of this curva.• ture must; be sub t:r acted f rom tha total time necessary f or the initiation of t he cur va t ure in order to f ind t he time required for t he inhi bi t or to pas s through t he section and arrive at t h e top of t he coleoptile~ From fi gure 22 t he total time required from the rnonient of appli ca tion of inhibitor to the beginning of the response in the coleop ... tile i a 62 minutes . Sub t rac·tinq; the 37 minut e s, as ex ... . p lained, indicates t h at t h e inh i b itor ·was tr ansported t hrough t his 4.2 mm . section in 25 minutes,. the rate of 10"1 rrnn . pe:t' houx- . This is at Caleulating the velocity from the second exp er i ment one f inds that i t i s 10,.9 mm" per hQu,r. The r at e of auxin t r ans por t i.n Avena coleoptiles i s be t ween 10 .. 12 mm. per hour so thf' t the value found in t hese t wo instances for inhib itor is the $a.me as t hat of auxin ♦. From fi gure 22 an indication of the transport rate of inhibitor through coleoptile sections in the normal posi-. tio11 may be determined. The 2 •.1 mm. fmd the 6.3 mi.11, show 54 . a de finite time for the beginning of the e u otoric phase . 'I'hi s t i me i s seen to b e 62 minute s fo r t he 6 . 3 mm. sec ... tions wh ile fo r the 2,1 L~a . sections it is 40 minutes. The di.fference be tween these t wo times, 22 m:i..nu tes , is the time requ ired f or t h e inhib itor• to h e tr an s por t ed thr ough the addi tiona l 4 "2 mm . of' the 6 . 3 mm. se cti ons . Thi s i s at t h e rate of 11 . 5 mm. per·. hour or approx imately t h e s rune as the tran sport r a.t e fo r the inverted sections . As this va l u e is based on on l y one expariment it s h ould b e bor ne in mind t hat it i s only a n i ndi cation of the trans por t velocity- of inhibitor in the norma l direc ti on . H. Tr anspor t of purified inhibitor in r adi s h hypo~otyl secti on s . An ex periment simi l a r to t he on e just de scribed for Av ena sections was carr ied, out using r adish hypoco tyl s ec ... ti cm s. These were 4 . 2 mm. long and were out f r om t .h e upper hal f of the hy pocotyls of plants thr:t h ad been grmm in t he gre e nhou se in the u s u al wa y . Fife di f ferent sets of t we lve plmnt s each were t e sted on the kymogr aph by the tec hniqu e previously described . 'I'he sets tested were: l . Sec tions in the nor ma l position with i nhibitor appl ied on top ; 2. Control s ections in the n ormal position with b lank ag ar ; 3. (• ec ti on s in t he inver•ted pos i tlon with in.. hibitor applied ; 5 , Inhibitor of t he same eoncentration 55. as in the 1st and 3rd sets tested directl:,r on the coleop• ti ".e . The re s ults of this experiment a.re g iven i n f i gur .e 25 . Here it is s een t ha t as with the Avena sections , the t ransport of the inhibitor t.1,.r,·,.,ugh the . inverted s •e e tions s hows at first a d efinite a u.xin c urvature followed by an inhibitor cur\ J. ture . In t his ca:::;e, ®os in the case of the Aven a, hydrolysis of inhibi.tor may take place at the cut s ur face . Fro-n this experiment lt ls also clear that ln a,:::reement with the earlier experiments it ls found t ha t in- hib5.. tor ha s r:i.o trans port polarity in radish. In t he one exceptional experiment on polarity of radish hypocoty ls .for inhibitor it will b e recalled that it wa s .foimd that dur•ing the two hours a llowed for trans - por t nothing but auxin h a d come throug h the i n v erted sec ... tion ... As may n ow be understood from f'i gnre 25 ,. t he two hours allowec f -n, the inhib itor transport was i n s ufficient time f' or inhibitor to come throue:h the section in l ~rge enough amounts to es c ape hyd.rolys .i s , according l ·s only auxin was found i n t he lowe:r plate . From fi gure 25, the r a te for inhib itor t:Nmsport th.x·our:;h the 4. 2 mm. inverted radish sections is found to be 63 minutes. o.f 4 . 0 m o This is at the rate h our, or only f.lbou t a third as i' ms t as in the Avena coleoptile. Frori1 a similar experiment w' ere indole - 3-- ti',cetic acid ( 850 g a'rr1ma per l i ter) was transported t hroue;h 4 " 2 mm. racUsh hypocotyl sections in t he normal positi on , t he velo- Ne gat ive curvature. 3 nun. Ir.ivert~d secto with b.L·. k a.gar . H Normc.1. l s e ct. with blo..nk a.ga.r 3 Inverte j s e ct. with inhibitor mi,o r 6 ruff: . Inhibitor · li ~ct directlv to coleo!.;o Positive curvature. Fig . 25. Reaction rate of twice decapitated Avena coleoptiles to inhibitor transportej through normal and inverted 4 .2 um•• sec1;ions of Radish hypocotyl. 56. City of shoul t r ansport was found to b e 8 . 1 mi.11 / hour . This raereJ.y be considere d an indic a tton of the velo- c ity as only four plants were measured in d e te1•mining this val ue. Sum1."'larizing the f'ac ts concerning the transport of in- hibitor t 1'. irough Av en a and radish sections one may say :thr:1.t : 1,. . I nhlbi tor is t ransported non - polarly . . 2. It is possible to obtain inhibitor a ppa:rently fre e f rom e.tL."{:i.n by the nrnv ers e Transport'' puri fi cation method so that n o neg ative cur-. vat 1.;ir e s are found when sueh purified a ..L"{in is tested on the lr~~ograph~ 3• The effect of unpurified inhibitor• miRy b e dup ... lic a ted by mixing auxin with puri fi e d .:.nhibitor . 4. 'I'he more di l u te the crude extrac t of inl:dbitor , t h e grea ter wi ll b e any :-1 eg~tive cur vatur e s prece d ing ~he pos i tiv e one s , 5. Inh1bi tor may l')e ~ydro lysed at c u t s u.rf' aces, 6. Inhibitor is t r ansporte d through inverted .a nd :normal Avena coleop tile s ections at the rate of ab out 11 mm , per hour . 7 ,,, It is indicated ths.-t inhibi :tor ls transported through inverted r adish hypoe otyl se ctions at the ra. te of 4 • 0 11'.U1! . per hour •. 57. Chapter VII . Physiolog ic8¼1 ho l e of' Inh ibi. tor. Since it we.s Vl'E:i.~ l l ests.b1ished t r.wt t h e inh i b itor sub- stance was esps.bl.e of beinr9, hydrolysed into auxin and ye t t hat 1 t coul d c a use gr owth i nhibi t ion in t he .Avena p lan t, it was interesting to inquire :into its po.s sib le physi olog i ... ca l role in t he radi s h pl ant . d ue tion , one c lass auxin . )f As po i nted out in. the intro ... inh:tbi tions arB known to be c au.s ed by The que s ti ons .novv raised were:... 1 11 Coul d inhibi - tor act the same way in these inhibitions as the: applied auxin: ; 2. Could t he inhibitor a.ct like an auxin precur- s or and be hydrolysed to y ield auxin in the plant? ; 3. and Cou l d t h e inhibitor wave a r•o:}.e as an inhibi tor :i.n the phy s :i.ology of' t h ® radis h p lin:1t? I, ~ Inhibitor d oes not i :nhib i t bud e;rmvtt·1.- !Jihe first prob l em lnvestig s.t e d of the knovm auxin inh ib itions was that of' bu,d grow th. 'I'his W9.s done in the fo llowing way : Ei ght day old eti oleted pea seedling s {stra in Alaska ) ·w hich h1;1d been ~srown in sa.:ncl, we:re cu.t off' near- t he tip just be l ow t he thir d node. On the cut sufi .. face of t hi r ty s nch stems purified. inh:'Lbitor i n lano lin wa.s s.ppli e d .. To a s econd group of tb.t rty stems inhtbi tor waa s.p ... plied mixed in t he r atio of two pe.r'ts of inh1b1tor- and one part auxin . 'l'o the third gr oup it was appl i ed GtS one par t :tnhib itor and 58. two parts e.1..1.xin. The auxin used ab ove was in a lanol i n paste in ~- concentration of 1 p~x t in a 100. A fourth lot of thi r ty pea stems were tre'"tted with the a uxin past~ full streng th, while a fifth lo t we:r e t reated with b lank lanolin. After s ix days the outg rowth of the la tera.1 buds was me asured. None of the buds developed except those oµ the stems tr e ated with inhibitor or t re Gtt,e d with lanolin alone . These results, g iven as t he growth of tb.e buds wi th stand. error, on treated stems after six days, were; inhibitor-... 87.7 1nm., ± 4 . 4 ; 77 .4 t 6,.1. control (lanolin only) ..,._ See also fi gure 26 whieh shows a toxic e .ffect of the inhib itor as evid enced by the a t rophy,ing of the tip cen timeter of t he stem ti ssue. From this experiment, which has be en repea.ted wi. th similsr results, it is a ppar- ent that t he inhibitor h as no effec t on bud inhibition. It might also be s~id t ha t at the concentrati ons of auxin u s eq inhibitor has no effec t in counteracting the auxin inhibition. B-. Inhibitor is not active in meso:phyl l growth. Since inhibitor was found to disappear during the growth of the l eaves i t was wondered tf it could function as a fac t or fo•r l eaf growth~ 'I'o se t tle this point Mr. David Bonner kindl y made a leaf growth test according , to t he method developed .by him, (Bonner, Haagen- Smit., & Wente Fig. 26. - Etiulated pea stem bud inhibition test ,using: Inhibitor; Indole acetic acid,and;Lanolin only,oontrol. Note lack of bud inhibition in A,~lso atrophying of its stem tip. - In.:iole acetic l:100 59. 1939). Unp1.11?ified inhibitor was used in this tes t . this metho d 5 m.rn. circule.r di scs o:C, 14 da-;;· f oliage . In old first leave s of radi.sh plants are f loated on a 1% s ucrose so lution. Appropriate con t.l•ols a.re run.. The solutions containing t he leaf' discs are pu t in an incubator a t 25 de§rees c. and allowed to grow for 30 hours at the end of which time they are weighed. The weighing is done in a standardized manner so that any increase in wet wei, ; ht of the leaves over the sucrose control$:; J.Q directly indi- cative of' the -a.mount o.f growth" A d :1. l u t 'l.on series of the unknown substance being tested is generally run . The re- sults of this t6st f or inhibitor showed t ha t the growth of t he leaf di scs did not increas e over t he su-c r·ose controls• It is therefore concluded that the inhibitor s ubstance under t hese cond:a.tions is not a factor formesophyll e;rowth, C. Inhi bi tor inhibits root growth. Since root growth is inhibited by auxin it was desir eable t o see if applice.tions of i nhibi tor would act , likewise,. For thi s experiment purifi ed inhibitor was used and the teehn ique used by Dr ., J runes Bonner was f ollowed. Jhelled Avena s e eds are loaked for two hours in water and a.re then la.i d out on moist filte r paper for twentyfour hours. At t he end of this time t he r a.dicle is ap ... I prruc.ima t ely ~ .. long .. From t he germinated. seeds those 60. are selected whose roots a.r e of a ppr Jxir:1at;e uniform leng th. They are t1>a11sfe1~r ed to Petr i dis h es containing filter paper and 4 cc. of the solut i on be:ng tested. Pifteen plants are put in each di s h which are then put in an incu.... b ator at 25 c ~ Twenty hours later the increase in th@i+" r oot length is meas ured . Inhibitor was te.s t@d in -the above manner in a: 1 , . l and . 01 di l ution series. In another simila1.. series If' di lutions , i nhibitor was hydrol ys ed into e.uxin by h E, ating the so l ution for 10 minutes in a bo:i. lint 1J:r..ter b ath. Du- plicate samples at eac h concentration of both i n.hib:i. tor and hydroly sed inh..ibl tor wer e r t.m . wer e also run . Water control solutions Standard aaxin and inhib itor tests were nade on the inhibitor and hydrolyzed inhib itor . 24 hours after t heir preparation , Daring this time they had been kept in an ice box ,. Tl-ie res u l ts of t h e c ompl ete test are g iven in Tab le II . Here it may l>e e en t h e. t the inh ibitor prevented t h e growth of the r oots to t h e same extent e.s the a . : ;.n f -r or:'! the hy1 .• drolysed inhibitor. The Avena test used s ubsequentl y to determ:i.ne t he streng t h of t he orig inal sol u t i ons showed t h at t he i nh:1.bi tor s olution contained a larg e amount of auxin although not quite as much as the hydro l y-sed inhibitor solution . T'ni s cou l d only be acc ounted for by t h e fact tha t pur:i.fied inhibitor is extremely- susceptabl e to hy- dro ly s is. In conclusion of this exper iment it can onl y be Root g:rowtll i,n t innib1tcr1 hydrolyse4 inhi'bltor, and.1 wa.te:r . Data 1e. the average length in mni. of 15 :roots per oen()entra tion '.teeted.10 All oonoentrratien.$ i':\:Ul. in dupliQate . Net g :rowtn over the origi.nal. lengtb (2 mtn . ) is underlined: . Clone . 17 . 7 1. 00 16.8 0 . 10 0. 01 11. s ll. lll. :i 9. Z l.5 . l -- l5 . 4- 13 . 4 l b. B l? . 2 15 . 2 61. se.id t hat und·e r. these c onditions the inl1ibi tor acts like auxin to c ause a.n :l.nh i bi t ion in t h e gr owth of roots,, Ther e is still a question as to wha t its effect woul d b e if it were pos sible to test it in a puri fi ed f orm without h8-ving it hydrolyse into auxin. D. Indole acetic ac id does not inhibi t r oo t growth b y causing an accumulation of inhibitor. Since t.he action of a.u..-;:in on root g;3:'ow t l1 is to c a.u se a g rowt h inhib ition it was s ugg ested t b.a t it mi ght b e pos s ib le tha ,~ t he auxin ia trans formed into inh ibitor in the roots,, The answer to this point was sought by analyzing indole acetic aaid in~:~lted r oots in t he following wa- : J>, lot of a pproxima tely 165 , 26 hour germi n• t ed Avena. plants were inh ibited in t heir growth b y planting them in .,. 5 a 10 )1. indole acetic acid s olution. A water g row11 eon .... trol was also used . 20 hours after the p l anting - t hey were divided into roots and coleoptiles an< were ether ex tracted a c cording to the method of Van Overbe elt ( 1938) . extractions were made in all cases. Duplicate Just previous to the extraction the me. te r ial was washed thoro ~h l y on a Buch.. ner ftmne l; fresh weight cletermin{Rti ons wer•e t h em made, The ether extr act was taken up in .3 ee . of hot 1}% ag ~.r . This was cast into b loc ks 1. B .x 2 x 2 mm. and tested on once decapitated Avena plants . After 90 minutes t he neg a- 62. tive curvatur es vie.re meas ur ed . See Tab le III . .Af ter a 150 minu t es , e.s the y h e.d not c h a nged a ppreciab l y ., n1es.sure .... men ts ra t t h i s t i me were n o t tak en.- From tl'd s d ata i t was s G2n at once t hs.t t he in 1.lb i t ed c ole optiles did not show the pr esence of inhibi t or bu t on t he con trary showed only the pr E~s nee of a u.x i n. Nearly twic e the amoun t of :a.uxin pe:r gram wei '.?:ht was fo un d in t h e i n.ri:l.b i t ed plants t han in t h e wa ter c ontrols, 'l1he roo t s of t he in:µi b i t ed pl an ts, however ., were only 7 . 7 mI11" lonts wh ile t h e cont:rolw were 14.3 nun, long or nearly twice t he size.. Thc::,ref ore t h e amount . of aux in per r oot wa s appro,~mately t he same in both the inh tb ited and t he non"" i nhib ited roots. The coleoptiles wer•e of a pproximately t he same len,,-,th €A l n this ca se , per eoleoptile ., t h.e amoun t of auxin i n t he inhibi ted plants was nearly t wice as muc h as t h e controls~ E. I nh ibitor is not ac tive i n root formation .. Since inhibitor was able to act like a1u i n i n certain instances t he question arose as to whether or n o t i t woul d act l ike a uxin i n t h e for mation of r oot primordi a. To answer this qu est t on t rie f ollov. i ng root1n 0 experimen t was done. Etiolated 8 day old pea seedling s , which had bee n rsrown in s and ., h ad t he termi n9.1 buc removed and wer e cut o/ f at the f irst n oo,e ( n ear t he base )• They were t hen soaked with Table III . Ether ex t 1·aotable a.l.l.Xi n present in the roots a nd ooleoptiles of Avenm. pl ants -inhibited i n their growt h by treatment witb •5 indo le a cet io a.ci d. ,lO M. Extract t aken up in . 3 co . l½% iiil.gar . Auxin g iiren .as @;egrees negat ive curva ture of 12 Avena test plants. All determinations in duplica.t e . W?- tei- 90.:n t .xo.J.. ifoots I ndole) a oe tio a c i d. Oo leopt i l.e 2. 2. l . . .l . . l . ' 1 .. Ooleo,Pti l e 2. J.. Roo't;s i n grams . 671 . 708 . 491 . 632 . 451 . 613 . 298 .2.aa Curva ture ll . 3 l3.6 l.8 . 6 l8.2 16 . 5 20 . 9 J.4 . 7 J,5 . 7 Curv . pe:r 16 . 6 20 . 1 38 . 3 2i . 8 36 . 7 34 . 2 49 . 5 55.6 ~ F1:esh wt . g:r;; ru vrt . Avg . Curv pe:r gr . wt . 18 . 6 52 . 5 63. their bases in water for four hours ~ At the end of thi s time they were put , with their ba.s es (bas al 1 c m. of the s -tera) , in i riJ'l-bitor so l ution. A tenfold di l ut ion se1"'ies ... 7 of i:nhibitcr was tes ted ( .from 1 to 10 ). Seven stem-s were used at each concentrati on . After 24 hours the y wer e t rans ... .fer-red to a 2% sucrose solu tion of t he same vo l ume as th.e inh i.bitor.. ( At this time it was noted that the stems wh · ch had b een in the fu ll s t rength inh..:.bito:r had bec ome f laccid , ) Eeven day s l ater they were examined for roots 0 1• r' 'J Ot format i on . No r oo t primordia were presen t in any of t e inhibitor concen trat i ons al though i nd.ole a c etic acid controls s h owed 'fl-ie sterns were then allowed to st&nd for a we e k ~rrany ., with · thei r ba ses in a. Vitamin B1 s o lution of 1 mg . per J.i ter ., At t 10 e nd of ~his time there were s ti ll n o r ots present on t he inhibitor treated stems where~s the indo le acet:te aci d controls had b ecome well r ooted ~ s howed no ro-c, ts . 'l''he water control s Al l of these tr ea t ments wer e carried out in t he Avena testing d ark r ooms ., From this experiment i t 1Nas conclude•d that with t _,.e unp·,r i fi ed inhib i tor t her e was no effec t on r oot fo r mati on of Pea stem c u tting s . F. I ::-1:.·libito:r inhibits seed g erminat i on. Another question wa s r aised a s to the action of in- ri.i hitor on seed g ermi n a tion . It is well known t hat atl." Cin 64 ce.n inhibit the g erminat:i.on of s e eds . To invest igate this p int w:t t h respect to inhibitor t l:':te f ollowing experi ... ment was done using pur i f ied inhlbi tor . T'nis t est we.s k indly made by Dr . James Bonne r wh o has standardized the procedure, In ma king this dete r minf?l.tion the pe rcen t ag e of g er1111 ... nat ion of t:)mato s eeds is used a s an indication of t ~J.e ef ... f eet of t b.,.; s olutions be ing tested.A 1I'he percentag e of g erm... i nation is comp2red to t1at ·1 .rith wa ter alone , T.h e techniqu e is t o soak toma.to s e eds for• 1 hou.r in wa ter , t 1e:n to l ay them out separate ly on .f'i.l ter paper i111.1aPetri d:lsh contain ... ing 4 cc . of the solution being tested. during ':-~-ii c h time they are kept in Af ter> 48 hours , n 1.ncub ator at 25 de .. r;r e cs centigr ? ,~ } the numbe:t'.' of g ermina te d seeds are c ounted and the percent ag e g errnine.tion c1et e r m:5.:ned ., In this experiment one such test was . made of puri.fie d ~4 inhib itor , one of indole acetic acid 10 M• ., and two du• # • plica te we.ter control tests . were 100 seeds used . :i.n each deter mination t here The purified i.nhi bit ::'.!' wes; n ot -!Jes ti<;d for its amoy.nt of inhibitor activity at t he conclusion of the experiment but in view of the former experimen ts where i t was found t ha.t i t ws s extreme l y s u sceptib l e to hydr•o - lysis, it ze0n.s 'J' f.H'Y- li ke l y th$t much of the inhib itor h d prob e.b l y formed a ux in. The re snlt:.s of this ex pe ri• ment s howed 665t> g ermination for the con'crols., 46% g ermi ... nation for t h e indole acetic ac id , and 46J6 g er mination for 65. the inhibitor. It is seen t hat the e:ff ec t of the inhiLitor wa s ex actly the same as that 0f tr.d ole ac e tic e.cid ., 'l?he se would b e t he expected res ul ts i f the inhib:l.tor had , as s ugg e s ted ,. l:1:ydr o l y sed i nt o auxin . Dr .. James Bon n er has r e ported ( unpub l ished ) the pre- sence of a s ub stance in canned tomato j ui ce that inhib its t he c; er minat ion of tomato seeds . It was accord ing l y of interes t to determine whether or n o t this s ub stance mi ght be t h e same a s the inhibitor. r_fo deter mine this point an e ther extra.c t:l on was made of 300 c c. o:f canned tomato j tl!.ice , 11he ex trac ti on was made ac cording to t he s tandard tec hnique fo r il"'..J.~ibi tor extr aetion . I t was t aken up in l i·% fl.g ar and te::1ted by the k:ymograph technique . Only a uxin curve :..:ir es we r e indicated a s a result of thi s test . 'fue inhibi t or of t oma to s eed germination is , then , certai n l y not the same ~rn t h e r ad i s h inhib i tor . G. Growth inhi bi ti on by inhibitor, in tissues o ther than the Aven a co l e opti l e .~ In anothe:<." expe rimen t des igned to inve s tigate t he res ponse of var ious kinds of plant tis s ue to inhj_b i t or , the tnhibitor was a ppli e d t o the pl ant parts unilaterall y in lanolin . The concentr ation of the inh i b i tor was sue tha t when it was a ppl ied unilaterally at the mi ddle of the c oleo pt i l e , a.fter 2 hours a 20 degre e positive curve.tu.re 66. was present.. Unilateral applications of this p a ste e aused growth inhibition responses f r om sue > di verse tissues as the pet iole of DryophyllL1m; tendrils st;enis , and petio les of e ti o• la ted and normal peas ; steus of et:lola.tea. Vicie. fabe ; and hyp ·1eotyls of' radish. In the case of the l3ryo_phyllum the t ree. tmen t was unques tione.b l y tox:t c s.s e.fter 24 hours the treated tissue was fla cc id broke off at t he s te.m. nd a few days later the leaf In t he other c as es no tox ic ef:l:'ec t we.s noted during the week following t h e tre tm.ent. 0 vations were disc ontinued after this time . Ob ser- Eve n thou~-r,h posi t1ve cu:rva.tures s.ppeared as a resul t of the inhib ition of growth at the site of a. pp1i catior of the inhibit Jr , the tissues continued to crow beyo:n.d t riat place. ·r'n.e concl usion indicated from the c .ove r esults would be that the growth 5.nhib iting e.ffect of inhibitor was not spec ific for Avena plants alone~ H. I:nhibi tor does not affe c t the · photo t ro•pic org eotropie res pon se of .Avena c ol eoptU es . Inhibitor was fou. nd not ,m arkedl y t affe-ct the res- ponse of Avena col eoptiles to ph)tot ,ropism, or g eotropism although further experiments on thi s point are neees.sary. This was s hown in the fol lowing- way . For the phototro pic :t:nvesti c atlon four sets of 12 :pl ants ea.ch were used , The plants "llver e 30 -u. t.a.11 at the ti:ue of the beg inn ing of the experiment . To the f:l.rst se t inhibitor in l ano lin wss 67. applied uni f orml y over t h e tip of the co leopti le two hours b efore exposing the co leop t i l e to lig ht . The se c ond s et was a contr o l for t h e f i rst and m s simila rly treated but with pure l ano l in 13 lone , The thi:rd set was t re att:. d in the s ame mann e r with inhibitor in l ano lin but it WEtS app lied just b ef ore t h e ligLt ex p osur e, wh ile the fourth lo t were controls for the third and were Rccording l y tre ...,ted 1 d th l anolin a lone . All four sets were t he n ex posed at a d is-"I" tanc e of 3-~ meters., f or 15 minutes , t o the radiation from a 60 W. Maz da la.rap . They were t h en returned to the dar k r oom ., m d t he curvature of t h e p l ants toward t h e s ide wh ich had ~-i een ex pos ed was me a s ured 90 minutes 1 &. ter., The c ur va.- t ures were : Inhibitor a:)pli e d irr.m1edi &t ely 9. 7 ± .o5 Lanolin control appl ied lmnwd . 7 ., 6 lnh ibi tOl' a pp lied 2 hours previ ou s '7 .. 8 ± .,.04 .± ,.04 Lanolin control applied 2 hrs. prev.s .6.4 ± .01 Inhibitor sa."ne cone . as ab ove app lied µni l aterully, c urvature e.ftAr 2½ hours mo s tufy inhibitor ~~ e 13,. 9 ~eotropic res ponse of Av ena coleopti le s after tre atment a simi h .:. r gr ot:,.p of f our sets of' six p l ant s eac h were used . Inhibitor in 1ano lin wa s a pplied t o one s et two hours before, and to a n other set jus t 68. i mmediate l y before exposing t he plants to the geotrop:tc s timul us .. Tl1e re gu lar Aven a h olders were t urned so that the long a:x.i s of the coleopti l e was pe rpendicular to the f orce of gr avity ,. The negative g eo t ropism resu lting from thi s stimul us was meas red af'ter 75 minute s. The en tire procedure was c e.r ried out in the Avena te8ting dgr k room wi th pl ants thnt were ab ou t 35 iiilll Q tal lo The g eo t r op :Lc cur -- va tures ~re"" s ur ed i:n the fou r sets of p h m t s .,.,ere: I:nhi.hi tor appl ied i :ru1ed iately l :3 .,7 ± 1.,7 Lanoli n app li e d irm.nediate l y 21.5 :l: 1.1 Inhibitor a pplied 2 hrs ., pr evi ous 21.3 ± 2.0 Lano lin appl ied 2 h o u.l" S pr e vi ous 24 . 4 * 1. 6 Prom t his data. 1.t i s cletirly see n t h at inhil)itor d id not e ffe c t either the g eo t ropio of the p l ant .. or pho t o tropi c res pons e Other experi 1en·t s have likewise inc1 icsted this conclu s i on ., I. Inh:lb i tor does not p cr ma ncnt l y stop c rowth . It wns al so of :l.nte:r·esv t::; inqu ire in to tho ca peclty for r;rowth of the pl ant after t he ~pp J.ication of ::i.nhi°'.) i t or . 11:'nis 'N& s done by a p p1yi n 6 i :nhibi t or :i:-::, e ce.r b locks once m-:i.d t wice decapi ts.ted pl ants . t :) bo th The de 6 r ees of posl tiv e c urvt:-1 ture nf ter 150 minut e s v-. ere then measur ed and t he ag ar l ocks r emovec1.., '11we nty fou r hours l s ter the c urvature 69 . wa.s b\gain me asured ;, '1'he p l ants we re kept i n the d ark r•oorn thr cmgh out the course of the e r,.pe r•i ment . The resu l ts Positive cui-•vRture , once deca pi ta t ed plants , 2½ hr s-., 24 .. 1 ± 1 . 4 f! V! fl tw:i.ce fl fl II 11 fl i 24 h-r s •, bac h vslue is the averag e of twelve i nhibitor tes t plants . It is see n the t in b o t h instances there was c,bout 10 degr ees l es s pos i tive c urvatur e af ter the 24 hour peri od! . This indice.tes th.:,it e;rowth h as taken plac e '.)n the side that wa s inhibited 21ter t 1e re:m.oval of the inhibitor even at a r:reater r ate than on the m,rmal side ., resu l t of g eotr opic stim.u la ti on as well This may hav e been the t: f..'l t he effect of a.n increecsed supply of food fe. c ~vhic b. hi:td not be en u sed du.Iwina: ,__ the period tha t the inhibitor was active . Tbis expe rlm$nt 8.g ain demonstrates th€>.t inhib itor io capable of :i.nhi:Ji t i n g g i-'owth end yet not p er manently stopping it .. J. f.;u .x:in-like ae tion of i.nhi bi tor j_ n the pea stem f!:,.'owth t es t . Ji ttention was new turned to the :-:.e tion of l nh:ib i tor i n 70. in the Pea test f or• g r owth promoting substances as g iven by 'l/ent !: Thime.nn (193 '7),. In t his test the amount of gr owth of the t h ird inte1"'node of etiohtt ed pea pl ants is us ed as a meas ur e of t he gr owth ac:ti v i ty of t h e s u.bs tance bei n 0 in... ves tigated . This in ter:node is long itudinall y s plit into two e qual h a l ves dovr:1- to within a ce ntlmeter of t 1e second node. It i s t h en c ut off b elow the second node and put in the s olution being tested ,. The gr owt h curvature of t he s plit i ntern.ode is manifested. by en inward curli ng of its tips ~ qeven spl it stems , per ~olut i on tested • were used. As es.c h spl it ste:r:11 had two tips t ha t cou l d eurve., this r.: a,r e 14 stem ourva tu:p,e :ne asurements. 1'l'le i:nw r d c ur vature is pro- pOl?ttonsl to the amount of r :ro.wth tha. t has taken p h1ce and is 11easured after 24 hours. It h as bee n s h own by iient {1939 } that certain compouno,s,, phenyl butyric acid, cyc l ohex r,m e ac etic acid_, ~re capable of c ausing a npre par atoryn growth :re ~e .t ion in t • e Pea Test . By prep&re.tor y ree.ction is meant tb.J?..t the ability ~f the ma terial t o grow i n response t o auxin is 1.ncressed efter m, trea t ment with these substanc es., Ac cordingl y i n tes t ing the purified inhibitor for its activity in the Pea test the comb inations of p:retr• t: atm n t and after treatme n t i n Table IV" we re ma.de . Her-e it is seen : L, f:S given That the in ... hib i tor v:as 1..rn ed for a pr eparatory reaction ; 2,. rrhet :l t vias tested on mi: teriel wh ic,.. h ad been g iven a pr e par at0ry 1 r e action ; and 3., 'l1l1<>t its ac t ion a lone was t ested"' The Et'feot ot purified inb.ibi t o r in the Pea growth test . The of' · inward cuiva:tau~e. Of the t ip~ ·. In pa re:nt.nea1.e i..s the. numbe:r of t1:ps showing :first number ie tbe average degl'teei no ourva.ture ,either inw&-rdia or 0.'1twa.rd•,.,..- j-,:ndica tive ef slight growth p i·omottng S;iiftivii.-tJ·. Tl1e underl:,tned numbe:r is · ·-• e :rn.un·oeI? of tips @Urving outward ~•- i ndtoa ti ve of no ·roiv ta act ivity . Pretre ated 2 hrs . Phenyl but yrio a.c1d I nhibi t or After .,. CO) 2. 1oe (0) Q 63 (O} l a 87 (0) ! 37 {O) - 38 (l) 1 28 (6) 5 Ind.ole a.oet i .o a.c id , '• 6 l'tl'i!, •· / 1 . 6$ Indol e !:i.cetiq 34 (a) a.oid,.• 2 mg ,. / l.·. Inhibitor " l.l (4) i 0 (0) - · l - 71. res u lts are shown in the same table . P:rom t his d ata.. it is seen that t h e inhib itor is ab le to e.c t like an ~:uxin., It responds to the pretreatment -. s does the auxin , is unab le t o g ive any pretreatment i t self under t,l,).ese eo ucti ·:ions,and ac t u ally cau ses a g rowth promotion. A kymograph analyses of i nhibitor solution af terward s showed that the inhib itor h a d been h;.rdrolysed into auxin so that its au.."r. in-like a ction was q:-1.i te und erstan .,_ able. K. Po s sib ilit,y of i nhi b itor acting like tatn a uxin precursor The n ext question was now as to whether or not in- t10 hlb i tor acted :i.n t h e r a dish cotyle dons as an inhi i tor or a s an au..~in precv..rsor h ydr ol y sini: :; t o e.uxin ,. oc en sh.own b y Van Overb eek (1933) .;ha t It h ss already aux:i.n diffuses ou t of t he cut snrfaces of the pet iole -s of the c otyl edon s.. It was f elt thu t if i~ were possibl . to d etermine whether thi s a uxin ·ere a uxin a or b , or indole acetic ac i d (which is pr obab l y t h e aux in from inhibitor ) lt would furnish evi• dence thE>-t this diff u sing auxin c ou l d or cou l d not come f rom the inhib itor, i\s the b est me1:1.ns o:f disti.nguish im.g b etween the a.uxins is b;l their mo lec u ls.:r weig hts , molecular wei ght determinations ~'1e:re made: for • t h e auxin diffusing out of t h e cut Burface of t he radish coty ledon petiole; for lnd.ole ... 3 - 0.cet i c 0.cid; H.1."'ld f or ;mrlfied i nhibito r. The 72 . molecular weights of inhib itor and of the aux in diff1.1aing from the co ty l edons were determined ~:n tI. ee separate expei-•iments . The me t h od u sed was that descri bed in Chapter• IV . In every determination a e,:raph of the coneentr· ti-on-curvature re 1.a.ti ons h.ip we. s made using two coneentratio!J'.':' c:.f' the cf t he substan.ce b eing determined.,,l and ½. This was done in order to be s ure of t he relative concen t:re. tion of the substance in t he agar plates , In t he inhibitor test this graph crossed the abscissa. at t h e orig in so th.a t the angles measured were a direct indication of the relative amount of' inhibit o:r in the plates~ The results of these determinitions are; g'iven in Tab l e V • Here it is r eadily seen thnt the evidence would indl~ . c o te th~t the au..xin diffusing out of the cotyledons indo l e acet ic acid . WS.$ The results obtained. here for the mo l e - cula.r weight of the :1.nhibi to!" a.re lower than t hose g i v-en by Mr. Hedemann who u sed t h e r.1a.rne method. ~rhe res. u lts in . TfJ.ble V . were ob tained ef'ter oone,iderab le practice with tbe t ech,;.'l'}_iq1.ie and. it is felt the.t the!r ere val id wi thi.n the l:l:mlts of the method. I t s h ou ld b e p i: :inted out that thi s method is not considered accura te to more t han 20%; because of this it is usefnl not so much for e stablishing the e.xact molecul ar weight of a s ubst ance as it is for indi cat ... ing substances of known mo l ecular weight . In this cas e Table V. Molecular we ight dete rmina tiona of; A. Purifiad inhibitor . B. Auxin diffusing from r act ieh cojyledons . o. In.dole acetic a cid . ( •n n is number t est p lant~ me ;-J,eured for e a.eh detern1inat i on.Fo:r method aee Chapter IV . ) A. Pu:r l f i .e d inhibitor . l .) Relat ive If Tot . :l.e 1/'l "x • cono . l0.,000. 10 . 2 3950 . l4l2 7. l 37 f>0 . osoo • "J: • 0., 18'00 . 1440 ;;$ . 6 1400 . l,l.95 l5 . l 3950 9.6 36 h time 41 min . Mol . Wt . 117 Temp • 19 . 88 mm . Avg . X . 12 12 D . 694 ~l.412 n 36 b . 98 rflril e 2560 . 046Q time 42 min ,. D e.822 8 .4 2200 . 0885 Temp . 20 Mol . Wt 5 .. l. 1340 .l235 Avg . X l 5. 3 3~90 . l4l.5 n 48 12 .. 1 3080 . 1531 time 42 8 .. 5 2160 . 095l Temp . 22 3. 4 860 . l7SS Avg . )t 2. ) • '.- • 83 ,. 0998 3. ) min . . 137 2 h l . 03 nun . JJ . 675 Mo l . Wt . 142 Table v. oont inued. . Auxin illtffusing f1•om r ad ish ootyledona . Relative Ii' Tot . is •x" B. ) cono . 10,000 . l.) .sa 10 . 8 3~~0 . 1459 n l2 8 .0 2i20 • 1lr3? time iO min . :0 5. 0 1820 . 1600 Te rt!p 23 3 .6 l3l.2 . 1274: Avg . X . l.36e. 10. 7 4020 . 1534 n:n 12 h . 98 6.9 2595 . 0540 time 42 min. D .741 5.6 2l,06 . 1060 Temp . 23 Mo.l . Wt . 104 3 .4 l.280 . 1300 Avg. ~ . ll.09 14 . 4 3532 . l.l.32 ~ 1.a h . 98 l.2 . '7 3l,l0 . 1641 t ime 40 min . D . 678 8.9 2180 . 0917 Temp • 22 Mo;!. . Wt . 4.8 1178 . l,402 Avg. X ~ . 631 Mol . Wt . l44 ~. ) 3") C• ) 125 . 127~ Indole a.eetto aoid . 1.) 14 . 6 3816 .1354 n ') ].; . t.# b . 98 ll . 2 2829 . 1137 tin)e 44 min. D . 600 6.9 l.SOB . 1109 Temp . 19.· D1 Theoretieal 5. 6 J.4aa . l038 Avg . X ·. . 56~ • l;}Oij Mo l . Wt . 159 Actual M.ol Wt . 175 73. it i.s elearl:,r s how.a that the the a ux in di f fusing f rom the co tyledons is neither e.i1xin a nor b .. Since th.e on l y other knoim na.ture.lly ocourr1ng ~mxin is in.do le ac e tic acid the c oncl usion may be drawn that the coty ledons is indole a cetic acid ,, atL"'{.in coming f'rom r adi sh I n Chapter IV it was p ) inted out that the differential destr uction test of the auxin obtained f rom inhibitor by alke.lin e hydroly sis :1.ndi • cated that it too was indo l e acetic ad.a.. Tm1s this sub ... sta.ntiates the evidence fro m the molecultllr weight determira t:tons and indicates tha.t t h e nt; t ura.11~ occurring auxin in r adish c o tyledons is indo l e ac etic aci d. From this dat• -chere i.s no reason to believe t hat this indole acet:l.c acid could n ot come from the inM.bi tor. L. Effect· of inhib1 tor on au..xin produc tlon in Av e na colepptile tips , and Radi sh cotyledons. It v,ras next thou~~t that if the inhibitor wer·e being hydrolysed to form. the auxin diffu sing fro m the radish coty .. ledons, as ind.5.cs ted , t;ha t it might oe possible to increase this diffusion by a ppl y ing in.hi b i t:or externally. 'l1o do this puri fied inhibit or was taken up in l anolin , which was then unifo rmly s pread over a microscope slide. Cotyledons from seven day old radi s h s eedl ings were he.rvested. and soaked in water .for ten minute~•.. The:-:, their upper sur f ace was 74. pressed d,o~m in t o t he· l anolin p aste on t be microscope slirie~ - 1½% ag ar b loc \ .s 1 x 2 x 2 mrn. were pn t on t he cut s ur f ,.. ces o:f.' the petioles and in thls way the s ub s tance out diffusinp.;/40 1u'.'in,5 the ns,rt. two hou.tas was collected . t hen anal y zed by the f vena tes t . I t was Control co tyledons were run a t the srune time b eing t reated. with pure l anol in al one. Durinc t he di ffu sion perioa , t he cotyledons wer e in Petri dishes l i n ed with moi st f'ilter p&pe:r. left i n diffuse room light . The dis hes were The ex perimen t was done in dup licat e using twelve c o t y ledons 1:n eac h ser l e$. 'i:he res ul t s were: ... Treatment Curvature ILhibi tor Control~ L~nol in only 18 . 4 F'ro:m this da ta i t :Ls s een thnt there was no effect of' the inhibitor to increase t he yi eld of auxin but tha t on the c on tr ar y i t de-cre a s e d 1. t ,., A simi l ar ex peri1Il.en t was tried u sil'ig the tips fr om 30 m.m. tall Av ena coleoptiles .,, In this experiment the t ips were cut of f of t he coJ.eoptile a nd kept on m·:;i s t fi l ... ter paper fo1• h alf an h our I' T'ney were then plac ed on l ·l% ag ar plates, 1 x 6 x 8 mm . and e ch tip t re "' ted 1;vith a 7 5. s me.11 dro p of' inhibitor i n l ano lin . lan olin alone was r u.n . A contr ol b rou.p using Both sets w~;re a l lowed to s tand on t he pl atss for '75 nunutes . At t he end of t hi s time they were cu t into 1 x 2 x 2 mm .. b locks and ana1y2:ed by t b .0 Ave n c1 test ,. '.Phe t•esu l ts ,, as clegr e e s of neg at ive cur vature per 12 test pl ants with stand ard error.t were; ... t r c Bted with inhib ito:i:• , 1 5 ,.'7.:i: 1 .. 2 wi~h l an olin onl y , 12 ~9 * . 9. Aven a tips Aven a tlps t r e ated As t he se res u lts are d e~ rived f r om onl y or:,..· experiment t:hey e a n not 'b e regarded a,s being e s tablished . They indicate howev er , that a ppl ic a ... tion of inhibit ) I' t o Avet1a coleopti le tips doe s increas e the auxi n; prodm.c tion , It is c oncluded then , t h&t i n many ins tan ces i nhi bitor e.xer t s physi olo~ t eal inhibiti ons 1.i ke iv.txin bec a us e i t ac - tually is bein,g hyc roly s e d into auxin . It :t s e_lso c;ono lu_,... ded tha t circunistent ial ev ide nce lndlcate s that i t · c ould ac t like an a u.,"'ti:n pr ec ursor in the r adi s h cotyledon s , 76. Chapter VIII . A_,. Mechanism of the Inhibiting Ac tion . Effect of inhib itor on food factor distribution_. According t;o Went ( 1939 ) there are t wo poss:l.b le mechanisms by whi c h a g rowth inhibit i on may come about .w 1i irst, the inhibitor may inter fere with t he growth process 1 i tself directly as seems to be the case for. the radish inhibitor , or secondly ; it may eliminate one of the sub ... servient factors necessary f or th-e growth p:rocess , Since the process of' growth is so li t tle understood it is useful to classify these se¢ondary factors under the g enersll h e ading of "Food F~c tors",, In t he ab ove r ef er~ ence Went has shown that ':!$rtain substanc e s , gamma phenyl bu tyric acid and cyclo texane acetic acid, are capable of inducing po s:t tive c t1rvatL1.res in less than 2½ hours in the Avena test by causing a. redistribution o.f these food factors. These positive curvatures are only found in the basal regions of' t he Averia coJ.eop tile , however, and are never found near the tip . Plants so trea.ted s how a negative curvature at the tip . This S shaped curvature is just the reverse of the S shaped curvature caus ed by inhibitor as mentioned in Chapter VI. Here the plants were curved negatively at the base and positively a.t the tip, Thi s d ifferent shaped S curvature is e.x.plained •by 77. '!;'!Jent on the fol lowiri..g basi-s .. 'l'he unilateral a.pplie$tion of these certain s ubstances , phenyl bu tyr:i.c. acid e,:~lo h e1ume aeetic, ch ang es the normal dis t ribution of the food factors so that they tend to ac ... cumulate near the tip of the ooleoptile, i.e. the place. of applieation, rrhls esuses ~ deficit; in the food fac - tors; for t he b ase and results in a growth inhibition and posit:tve enrvature at thi$ place. 1.Ie~r t he tip the opposite phen.omenon occurs e.nd here one finds n surplus of food 1.'e.etors and a negative curvature. This e.xpltan~• tion was proven by t he :following experiment. Avena coleoptiles were treated at the tip with le.no ... lin pastes of' th.e subE;tances causing the food f'aeto11 :redi s ... t ribu tion. After two hours, during wW.ch time c011.trols- with blank lanolin had also been tre a ted, the plsnts we:re decapitated and cut into 4~i2 mm. zonea which were kept in t h e srune sequence as they occu r re·a .Ln the plru-it. rrhese consecutive zones were t hen put into s. 1½% sucrose solution for 24 hours and at the end of that time their gr owth in leng th mea sured. with an oc u lar mieromete:r:-. In the un ... t reated plants the gr eates t amount of grewth v1-a.s found to ~take place in the second :and third zones·s with still con• si derable growth. in the fourth zone. With the tre a ted plants, however, t he f'irst two zones showed nearly all of t he gl'oWth; while the subsequent ones grew only slig htly"'· This indicates that t he . treatment h ad eaus ea. an ae c u111u ... 78 . u le.tion of the food facto:rs in t·h v upper par t• o,L "" t he co l eop.,.. 0 tile and a d eficit in t h e lower pmott am.d t h u.f fU ld rWbY:fkht ab out a positive curvature in the lower part and a neg a .. t ive cur vature in the upper part ., It was of i n ter est to see if e.ny food factor red:ts ... t r ibution occurre d. -.-,i th r ad:i. s h inhib i to r~ The same ex peri.-. ment was per fo r med as descri.bed ab ove using two concen... t rations of' inhibitor,. concentration . F'ifteen pls.nts wer e 11aed at eac h The de.ts. are given in fi g 27 " The experi ... men t he_s been repe:;i.ted three- times \1ri th similar re su l t s~ Fron1 this graph it is c le arly seen t h at t he i nt l.ibi tor sub ... stance does n o t a.c t by c ausing a :redistr i bution of t he food factor. These res ul ts would h a,re b een ex pected. f rom the differ en ce i n the type of S curves caused ;Jy the inhi .,. b itor and the aforementioned s ubstance s . It indice.tes a.g a i n t hat the inhibiting ac ti on o f the i n hib i tor i s on some phase of t h.e primary growth process its e l f' 1,ather than on some of t he secondary factors f or gr owth , P. s econd possible hypothesis for t he acti nn of the radish growth inh:lb i tor might be bs.s ed on the ass umpti on that the i nhibitor i teelf coul c ac t ' as :,recurs or of e.1-u::in .. ( ' ee a l so Cha pt . DO Under ordinary c onditions t hi s pr e ... cursor c ould be changed into auxin but when applied to t he plant tissues at an abnormal l y hi gh c oncentr a tion , as in this c ase , it would be unab l e to be conver ted , would a.ccumu.l Hte• 1n t he cells and inhibit the nor mal eC?rowth length after in 1½%. sucrose. - ·- ·.-·-1 Coleoptile treated 2 hours with: ! _____,,,.:::-:-::.::::.7-._ .-·-, Inhibitor ,full st rength · ,. ....... ...... ..... , blank lanolin. t ........ ........... J Inhibitor, . 3 full strength r·canirol 4.2 •• 0 3 mr. . • 3.3 mm 7.2 11.4 15 . 6 I I . I I I 19.8 17 mm . to coleop. ba se. A '<-• Zones tested . on Avena coleoptile. Fig. 27. Food f_actor dj,stribution in Avena ooleoptiles as indicated by increase in length of 4.2 mm. sections. Daah-dcit line full strength inhibitor, dotted line .3 · of full strength of inhibitor , solid line blank lano l in control. For full details see text. process.. The conversion into au.."'{.in _mi e ht be vis ualized s.s taking plsce throug h en zyme action. Inamnu.ch as the indications of the chemical structure of t h e inhib itor mo lec u le ·indio8tes the.t it is an ester, With auxin con st:t ... tuting one part of' the es ter linkage, this proposal bore f ur ther consi derat ion . B.. Action of' li pase prepi;trations on purified inhibitor ... It was felt that if the :tnhib ito~ mi ght be converted by enzyme action into a uxin , poss t bly such an enzyme woul d be a .l ipase.. To inv estig ate this :pos s :Lbi l ity a li pe,se pre .. paration was made f rom castor be an endosp·er-rn according to the method e;iven by Long enecker & Haley , (1935) . , The same tec hnique was used for the preparation of a presumed lipase active f raction from :radish seeds . The essentials of' t he technique were to grind the ungerminated seeds as finely a.a possib le and t hen to extract them wl '~h low boi ling petrol ether ., (Bo i ling p oint 20 ... 400) ., Tl'1e ether was then removed from t he extracted material , which was dried and ground toe. fine powder. This preparation from the :radish seed will be r eferred to as the "radish seed 11 preparation wh i le t h e car1 tor bean seed preparation wi ll b e s poken of a s the 11 1:tpa.seu pr ep ru."ation ,.(Activa ted at pH 4.8) To test the radish seed prepar at ion :fo::r its activity 80. in converting the inhibitor into aux.in the f ollowing e:x:periment W8.S per formed .. To three t e st tub es esch containing the same con.centre. tion of rn1rified lnhib i tor , ( suff'icien t t o c aus e 1 5 d.egi•ees posl tive c urva ture), t he fol lowing s ubst -- nces were added : To t h e fir st test tube was added 2 .., c . of water and 100 mg . of radish seed pre psration; to t he s econd wss acded 2 cc. of water but no radi s h seed preparati on; to the thir d was a.dded 2 cc. of water whic h con tai ned 100 mg . of t h e r s.dish s e ed pl'.'e pra.r a. tion tha t had. b e en heated f or 10 m.inutes in a boi:J,1ng wat · r bath . tub e \1as To a f our th test added 2 cc. of ·w ater end 1 00 111.g. of radi s h seed preparation but no inhib itor. put in an ino.ub e tor at 3'7 All fo ur test tu.bes were c. for 18 hours- i',t the en d of this time plain 1½% ag ar plates (1 x 6 x 8 mm. ) were soaked b 1 the solt1. ti ons e.:nd after tb:ree hours they were cut i n to smaller '1 loc ks 1 x 2 x 2 mm . and the growth activity deter mined b y th.e kymograph an alysis method . The res u lts are seen i.r1 fi gure 28. 1Iere it is clearly see n t ~1at t l1e presence oi' t h e radish s e ed preparation pr even t ed t he i nhibitor f r om chang .. ing into auxin instead of caus i ng i t to change as it was expected to do . This experiment has been repe ated thr ee times with t he sa....11e results tt A similar test was made using the lipase prepare. .. tion. This pr epa.ration ., as with t h e radish preparation, showed no effe ct of increasing t b.e h ydroljrsis of t he i n- Negative curvature. 5 !illi, • 2 mm. alone . itor plus boiled R dish seed prepn. mm. H ----·~adish seed prepn. -...._ alone. ·- -. iibitor plus Radish eed prepn. ,no boiling. Z mri.. Positive curvature. Fig. 28. Curva ture rate of twice decapitated Avena coleoptiles to: 1.) Purified in.hibitor a.lone; 2.) Purified inhibitor plus boiled Ra.dish seej preparation; ;:i.) Radish seed prepdration alone,and; 4.) Purified inhibitor. plus Radisn seed preparation, no botling. All four solutions kept at 37oc. for 18 hours prior to testing. Each point is the average of 12 plants. 81. hihitor, In fact as in t l1e radish see-d prepM•ation, ,jttst the contrary w. s :foun d to be t he case . In t he second and third test; tub es Wher e t h e ;Lipase preparation had been s.d- ded , ther e Wt s strong inhibi t or Rc tivity , {although in the t hird test tu.be where the li pase prepare.tJ.()rt :t).e:d been heat ... ea. b efor e beint, ada.ea. ,. the:re was les s a c ti vi ty than in the second i;est tub e wh ere it was added wit h no heating ... ) l'he first t est t ube , ho1ueve1... ., s howed n.o inhib i tor activity . '.Phe cont~ol on pure lipase prepare.tion alor1.e, likewis$ c uused no cu rvatur<:is . rh0 c oncl usi on is t:L ~ t 1 t he presence of' the ll pa.s e pr oparation preven ted the hydrolysis of the :t nhibi tor • c. Pr evention of inhibi tor hydr ol ysis by :Lm.puri ties in tbe extrac t ~ It h as also been found t ha.t when t her e are nmny- im... pur i tie~ , ch lOJ:~o pb:yl:}.., etc . in the crude extract, the i nhi b itor :mo lecule is not read ily hydrolysed~ whereas when these heve been. removed lt b.ydrolyB es very easily . 11:1.is w~ s s hown by the .fo llowin g ex periment t il1he crude inhlb i tor extra.ct WB s prepared in t he usuHl mim n er f rom :i:· adish cotyled ons ar, rJ on e v e,porating off the ether ..,a syr _u py gt·een mass of chlorophyll and f-; :tt;y s ubstances waf. obt ained slong with the inhibitor . v'les div ided- into two porti ons. This extract One por ti on wa.s ke pt in the ice box ove:rnight while the other port ion was heated i n a boiling; water b ath for 10 minu tes H.n d then kept at 3 7 degrees C. unti l t b.e next day . Both solution s were t h en tested on the kymoe;raph mac h ine , 'l' 1e results, f i gure 29 . showed the t there had. been no h ydroly·sis o.f the in- hlbi tor in either case . 1·he sa..me type of experiment us - inc pnrified inhibitor , with mo s t oi' the extracted im.plu:-ities r emoved , s ows however , complete h ydrolysis . ,)ee f:t gure 2i3: - 1 . It is s uggested t ha. t both in t his ex peJ:•im.en t and .i n the experiments und er section B, t l.:., inhi)itor was pre ... vented fJr>om hydro lysing by being adsorbed on t h e sub- stanc es added to the inhibitor sol u tion. In this l ast experimen t t he.s0 sub st -nce s vould be the extr ac ted impuri • tles ♦, D., Conclusions Fr om th.ese l ast two sets of experiments the indica'"' t:1.ons ar•e thot there i s no lipase .. like enzy-me in the r'edish seed which hydro l ys-e s the inhib itor and t hu s forms a.1.min . Moreover t hese e·:per im.ents e; i ve n ei t 'i·1er an insiE;h t into the mechanis.11 of growth inh:ib i tl on n or an e :-::p h,:nat:l.on of the action of l arge amounts of inhib itor in influencing any specific p '1!UH~ of the gr owth proces s ,. I n v :tew of t he f act that it vms :round t hat inhibi tol"' does not c ause & redistri • Fig . 29. Reaction rate of once decapitated Avena coleoptiles to crude inhibitor extra ct boiled 10 minutea,and with no boiling Each point is average of 12 coleoptilee. I 4. Hows mn1 . Boiled 24 mm. 7 mr11 . ositive curvature. . • -------· ~.____-Non-boiled. 83. buti.o n of t1e fo od factors one must be lieve that it acts by affecting the growth process di:i:•ect ly. 64. Chapter IX . A. Discussion and Conclus1ons a Discussion .. F'r-om the pr evious wor k lt :t s seen tha't t h e inc.J.cati ons 1-1r0 that the inhib itor., as it occurs iri the rnc1i$ h plant ., Hcts in n o mann er as an inhibi tinG a g ent . It a p parentl y onl y has t he proper ty of i nhib iting r r owth w·en it has be en extr a cted f r om the pJ. ant anc coneentre.ted to rrm..riy time s its n nt ur a l c oncen tr~1 ti on . In s pecul a tinc as to wh at .fa nction , i f a ny , tha t t h e i nhibitor subs t ance might h ,-ve , one wou l d t hus r u l e out t lrn t of inhibition . Since , however, :it Wi'.lS !'ound t hGt it ls readl l y hydr ol ysed :i.nto a ux in t his rn.riy b e an indicati on thnt t J:1e inhi bitor can fun ct i on as a n a uxi n pr e cursor . l t must b e b orne in :, ind tha t t h e au.Ein f ormed from :tnhihi tor is pro ,Jab l y indo lc acetic a cid and n o t aux in a nor b . A di s c u s sion of the p:res e n t s tatus of t h e wor k on a.n auxin pr ecursor is e.;i v e n i n Phy t o 1.or mones ( 1 9 3 '7 ) . He re it i s p ointed out that evidence indic ates t her e must b e an a i.t.'tin pr cc ursoi" wh ic h i s ab l e to b e stored in seeds , , an d mov e thr ouc;h t h e ste;:u act1op e .. tally . A sugges t ion i.s made that this pre cur sor m:1.g h!;; 'b e an e ster o.f a ux in a inas muc h as such esters are i n t ,_em.se lv es inac ti ve and yet could c onc e i vabl' be hydrolys ed 85. in the p l m1t to f orm auxin . 'l'h.e pr e sence of such esters in plan ts is a l so indicated . When ., however, esters of a uxin 0 are ac tua.ll y tested f m.- gr owth a.c t i v i ty t h ey eli cit no r e s ponse . Esters of :tndo l e acet i c acl d on t; i,1e other h and are ab le to underg o hydr o l y s is and do cau s e gr wth promotion . Inhibitor has been seen to paralle l the re q;_1 irements set fo rth ebove for un aux in pr•ec ursor . It is stored in and l s able to move seed s, throug h stem tissue ncropetally . lt is also an ester w ich ia readily hydrolysed in vitro into auxin. seeww to have t=i fair l y wide dis tr :lbution. . not 8. }rec u rs or of auxin a ., lt l t is , however ~ At this po:Lnt it may ·be well to p oint out t hnt wh ile the present evidence in i C:-:ltes t h • t au xin e. :i.s the growth hormone foune i,n bi gher p lants vhe a ·· ount of evidence is· rath er g e nerali zation., Further more t h e e.x_pcr :i.me:n ts presented in this paper· show t ha t the a mtin di .f fu sing from t h e rad :i.sh cotyl edons may b e ind ole acetic acid . Ag ains t the view... point t hat inhib itox- coul d act as pr ecursor of indo l e aee,.. ti c acid il1 the !'&d i sh p lant is the f'aet t hat when E:.sters of indol e -cetic ec i d ar e tested in the Av e na. test they --i. r-v e never been :r~corded as catrning any positive curva- 1 ture s . 'fhe se exper iments were n ot c one , howev er , with a view toward ob servin g po sitive curvatur es. In fact it (1936) that , wh i l e not of in.do l e ac t~t:tc acid , i n : oJ.e ethyl a mine at a concen tra-t ion of l mg ./ c c~ is capable of ea.u sing positive eu.rva- 6 ,1 hours . I n 18 hours t he curvature was still 6 . 0 de c;rees . Lower eoneentrations ( 1 mg , / 3 cc . ) fi rst caused positive c urv utur es of 2 •.9 c: egr e es in 1. 9 honr s , w::1.ich b ecame nog a..... tive cur-vr.;;tures of 5 .2 degr ees after 18 hou:r•s. Tb.i s reap• poarance of a nega tive curvature was not fund wi t h in~ , i b itor when the inh i b i t e d plants were observed 24 h ours after t heir treatme nt. This may have been due t o the fact that t h e inhi b i.tor conce n tration wa s so hi gh t h ·t t h e plant wa.s unab le t o fo r m suffic ient auxin to ov ercorile t h e inhlb i t.,ion . 'J.lhls s ame effect m.ay also be seen in · the above example of t he positive curvature with +-he h i gh conc e n tration of inc o l e ethyl e.r., ine . r urther g rowth curvat ure e x9cin"' i ments should be per formed w:1. th .. Jre dila t e in. , ib:ttor con cen trati ons than t hose used in Chap tor VI~ a.'1d ob served over a long er period of t:trae to Jee :Lf the nee: a tive c urv a t ure s found b y· Skoog f'o:r indole e thyl a .ni ne mi ght .l so appear with inhibitor. It is safe to say tha t f r om t he evicence pr esen ted in this work t h r" t inhib itor c ould f.!Ct as an auxin pr•ec ur• sor bu.+- 1nt i l furt:.1er evi der1ce :i.s obtain.ed , even t his point wi ll have to remain an open quest ion . 87. B. Conclusi ons . F'rom the exper imen ts described in this work the conclusions reg arding the inhibitor are: 1. That the re i s an ether e:i::tr act ab l e su.b stance in the cotyledons an~ l eaves of radish plants t hat '.ts capab le of causing posi tiv e Avena c urv atures s.nd growth inhib:t ... tions . 2, Chapter r . That it i s possible to anal y ze for t he amounts of tb.is sub stance on o. quan ti t a ve bas i s usinr; t .1e standard inh:tbi tor test . Chapter II ... Tl1at the occurrence .:.: ' t h is tnhibi ting s ubs tance i s 3,i not r•estr i ct ed to t h e radish p lHnt but t hst it is f'm.m d i'l o t her plants as we l l. 4 .. c ·_apter I1I . Tha t with the d r owth of radi s h le a ves their inhib i ... tor c nten t decrea ses and is f inall y a pparent. y abs en t be .. in 0, 5. repl e c.ed by nu.:.1£1'1:1. Chap t er I I I. 'Inat it ~as n o aci clic or b as1.c groups " ,ld can b e hy ... drolysed to yi eld a uxin . 6. Chapte:r rv . That it is probab l y-nester of ind ole acetic ac :J.d with s ome un1movm fract i on . ?• Chapt er IV .• T' at t h e positive Aven a c urv atu.res mtiy be comple t ely expl ained on the bas :ts . f the amount of growth in.hi) i ti.on c eus ed by the inhi bitor . 3 ._ Chapter v . ~Phat in t he unpurified c ondition i t has no effect on the zr owth of sections of Avena col eoptile a.nc radish 88 . hypocotyl . 9. Chapt er v . 'Tha t i t h as no po l a r i ty of mov ement: in the rad ish or Av ena plant . 10 . Chapter vr . r.l'b.a t it is pos s lb l e t o r emov e conta minat i n g au.x i n fr om t h e c r ude e x t r ac t by us e of t h e purifi c ti on me t hod . I 11vers e Ti-•an s port 1t Chapter VI ., Th at i t may b e hyd r ol ys ed at c u t surf ac es to f o1·m 11 . aux i 11. 12. 11 Ch a pter VI . 'l'ha t i t i ~ t r ans por t ed through Av e na c oleop t i l es e t a ppr o~imately 11 mm. per h our , or t h e se:,.e :r a te as auxin .. Chapter VI . 13 . Tha t it does not c ause gr owth i nhi bition a s a r e sult of fo od f a c tor r•e di s trlhbution . 14 . Chap t e r Vi!!. . 'l':ha t .:.t :ts po s s i b le t o pr eve n t its hydro l y s is in t o a u..."'C i n i n 'li ·ater s olutions by ·he aa d l t:l. 011 of i mpuri t i. es on wh ich it a ppsren t l y may- b e ad sorb e d . 15 . Thst in t he pur i f i ed condition it is reac i l y hydro ... l ysed i n wa t er. 16 . Ch a pter VI I I ., Chapter VIII . That i n many te s ts for phys io log ic al ac t ivity i t b e • hoves li k e aux in , but it d oes n o t t1ct li k e a.v.xin and caus e bud i nh..ibition 9 Chaptc:r VI I . 1 '7 .. 'rha t its mol e cu l ar wei gh t is poss i b l y b etween 100 and 17 5 .,. Chapter VI I . 18 . 'I'h et i t a ppe ars t 1.~ t ·· h e l, y .r oly s i s of' the i nhi b i t o:r :..n the r a di s h cotyl edons cou l d :1.cc ount f or t h e pr esen c e of aux i n di f f usin g ou t of them, Chapter VII , 89. Literature Cited Bonner,:O . M. ,A.J-.H.aagen Smit,& F . W. Went . li39 . Lea.f growth facto;r.~ I : B:i,,o- ass-ay and source f o-r leiaf grovi•.,h factors . Bot . Ga z . ln press . Bonne:r.-,D . M• • & A. J . Ha.w6 en Smit . 1938 . t,e.a.f growth fa.e t ors II : Actl:vity of wre substances in. leaf growth . Proc . Ma.t . Aoad . Sci . In press . Breejt,F . );1936 . D~r Einf lu.ss von Wuohi&toff""' und Si.urepaisten ·;; auf {~as Wei.e.•run ven Avea~~ und el: a :h.u ... keimlingen und. seine Abh~ngigke1t ·voni Sa.ueretotfgeha t der Luft . Jahrb . -w1ss . Bot . 82 : 5$1-612 . • • B:ruus,H . .R . li322. Dif'.f'uste3eilie1d en Vieoos.:iteit . D1S$ , Utrecht . ' Ozaja..,A . T;ti . 1934. Der Naqbvu~ i .a dej Wuehsetoffes be1 Holzpf lanzen . Ber . d . B,9t . Gee . 52 : 267-271 . ik .• Goodwin,R. H. ,,,aa ., Evi~ence for the pre s enx,·e tn oerta i.n ether ext ra.ots ot eubatanees partially masking the a ot 1v1ty of auxin . Am . J . Bot . 26 : 130- 135 . G<:>rter,C . J . 1927 . Ove:t het voo:rkoUien van groeiversnellende en groe1ve:rtra.gende. atoffen. . P.roo . Kon . Aka d . Wetene~n . Amsterdam ~Hh4li""423 . Kog; l,F. ,A . J . Haagen Smit,& H. i ,r xleben . is34 . froet den Etntlus s def Awc:).~e a:uf' da.a Wui-zel,wa obet1;m1 und uber cUe o:nemisohe Na.tu;· des A't{lX.ins der Gra.skoJ.eopt tlen . XI I Mittei.lung . Zei t . phyet.o l . Ohe?f.l . 228 : l04-l.l2 . Laibach,F . l.933 , Versuohe m1t Wu.ehsstoffpaste . Ber . d . bot, Gea . 51:386..-392 . Meye r ,F . 193($ . Uber die vertei.l;un-g d.es Wu¢)he,etoff es 1n der Pflan~e wallrend ihrer Entwl,o.kl,.ung . Diss . Frankfurt ,. Niel.s e n.,N . l.824 . Studies Oil the t.r a nsmieeion of stimuli ;J;.n th.e oo,leopt~le of· Are~~.. Pa.nsk~ Bot . Arkiv . 4 (8) . Onolm~L . W. l 9l.2 . Die tre 1e .Dif fusion der Nt.ehtel.ektrolyte . Meddel . fran Vet . Akad . No'bel.inatitut lad. 2.,No . 23 . 90. Overbeek,J. van, J.933. Wucb.setoff ,L1ohtwaohs t um~u;ea.kt.i on, UJld. Phototiopismu.$ oe:i Rap•an,ui>. D1ss. Utre¢ht. Overbeek,J. vaa,. isia. Auxin d.if.itributioa in eeedlings. and 1 ttl be~r1pg on the problem o.f oµd ~nh1b~ tion. . Bot. Ga.z. 100:.133;..J.G:E>. Ove:rbeek,J.v~., l.938. A simplified method fo.r a.uxtn extrac t ion. Proo. Na t. Aoad . Sci~ 24:42~46. Overbeelt,J.van, l93i. Au.xin 1n 1na.rine a.J.gae . Plant Physiol. ln preaa . Schei'fe:r and Ka.\Va.lki. 1923 . Handbll.1.0A de1; Physika.lis ech= Cb:einisonen Teennik by K •.Arv-elt. Publ:i.ahed. by F.Eldke, Stuttgart , Sohnei.atei.,C.L. a,nd F.W.Went, l.938, A P~i;tok ym¢graph metnod for the ana.lyeie of '.;he Av~tr tes1:tt'i. ·Bot . Ga..z . 99: 47·0 -496. Se ubert ,E,. 192£.5. ~er Wa ehe t wn$reguJ.a.t oren :Ln de~ Kole opt ile ven AYe§~• , : - ., . Ze:i.t . fur Bot . 17;4.s.-ea . 1936,. Some physiQlogio,al fune t ione, <af t he g rowth horrnone in higher plants . Thesis,.Oa lif. I nst .of Teoh. Skoog , .F. Skoog ,.F. la3'7. A deseeded )\ve:na t e$ t method for sma ll amount s of GL'Wtln and awt1n preour.s or e. J. gen. P.hys1ol. 20 :3ll•3 M . Starlt ,P. 1921. Stud.ie» Ube r trawnatotrope und baptc;>t:rope Re t.~le .1 tungevorgange mi t beeo,ndere;r; Berij.oks1,o 4t,itt;ung a.er Re1zubert:ragung a\lf fremde A:rten und Gattung<tn. J ahrb . wiss. Bot. 00: 67-134.. Stewa.r t,.W. '· , . w. Berg;ren 1 & C.Redemann . l939. A plant growth 1.nhib:i tto.r, Sc1etu:,e ss :.185,.,,iSa . Utsprung,.A. & G.BllUB. 1924. Elrie Meth:od.e zur Messung de$ \Va.n4... und. Turg ordruc:kes <ler Zell.e ,nebst Anwendun.gen . Jahrb. wiss . Bot . 6i ;l..,.ll0. Vietmiann;H, 1938. Untersuchungen iiber die ohemotropis cne W1rkung o.rgani .sehet s ~11.••~~,J Jahrb. wias. Bot. 87: 408- 4~6 . 91. VJe i j , H. G. van der , 1932. Der Mecban1smue des. Wuoh&eto:fftransportes . Diss. Utreob.t . Went,»• . w. 192? . Wttol'iaetoff und Waelurtum , .o~sfl . Utrecht . \'Jent .F. w. &. K. V. Tnl.im~nn . 1937 . Pb.ytobornione~ . Maom:1 l.l&n C0n1pany,.New Y:orlt . Went,F . W. & Ralph Wnit.e . 1939 . Expeximents on the t:t>ansport of ,ra.uxin . Bot . Gaz, ·100:46fi•~65 . Went ,F. W. ·I J39 . }.e.ase ef oei•:relative bu.d inhibition i n plant s . I n p;resa. Went ,F. W. l S39 . aux.in,. • Fui·ther analysis of the Pea test for • In press . Exte:ns1b:llity of Cell Wall Material in Indole-3wac eti c f cid. 1. Introduc tion Since 1931 when Heyn ( 1931) pointed out that a uxin :tn.creased t he exten sibil:l ty of the cell wall ( i n cludi. Lg ;10th p l astic &nd elas t ic exte nsibility) , t he ques tion of t h e mechan ism of its action h e,s been the center of mu.ch di sc u s-, sion . Two viewpoints on this subj e ct a:r·e possib le: Eiith er thct t he s u.x:in acts d i r ec t l y on ·'..;he cell wall or t ha t it ac t s i ndirectl y t ru:•ou.gh t he cytopla.m . 'l 'he evi den ce fo1• t h e l s.tter vievipo in t is well summarized 'by VJe n t a nd Thiman:n ( 193'7 ) • On the o ther h a nd , Robbins and Jackson (193?) performed e.~peri - :nents which su.r:;g es t tha t t he acti(!) n of e;rowth hor rnon-e is on t he cel l wal l d irectl y 4 In t hese exper ime nts it was fou nd tha t when ., 2 per cen t indole ac.etl c aci d in l an olin wa s ap-... pl ied to l :lv1.n g or d e ad cell wall rnrit eri a ls it c aused an inc re ase in t h eir e.xt~ns i b ili ty. It iivas also f o un d t hat t he same t r ea t men t dec 1•1_:.a sed t he extensib i li ty of b oth f r esh and dried root ce11 wP ll materials . Howev er, they pt>int out that t h e effec.ts ohtaine.d are no t n e c e ssar i l y spi::.c.1fic f'o:r t he gr owth hor mon e u sed ( indole ... 3 - a c-etic aeid ) a s n (:i . con t rols of pH were used; and f urther more, sine E?J the w,a.t.""r content was not lmown to be t he same in t11.e lanolins u sed , the resu l t s attr:lJrn ted to g rowt h hormone may have p a.rtly b -~en due to suc h d i f ferences~· In view of' these circ umstances e.nd bec ause of t he impor t ance of t heore• ti caJ. conelu s ions reg f':.rdin 0 gr owth mechan.i sras whic h mig_1t be based on s uch d ata, the ex pe riment s were repee.ted 2. with as neerly identical n1Hterls1 as possible . experiments were ~eve:ral new lso devised., E...xperi;nents AWD Results. -I . i]:'welve ;;,~ecee of 1,o . 8 cotton throt,d e.pproxi!;.utely 2100 rm. lon~ were suspended in a d&rk roo!!l w1 th e. 120- r;r am wc1e;ht e.tt ched to each . Three pieces ,;1ere tmtrE ate , three wer,, rubbed with ltmolin n l y , three wl th . 2 per cent indo l e - 3- acetic acid in lanolin , end three with . 2 per cent acetic acid in l anolin . Pour pieces of he~p , uppr o~im~tely 1000 mm . long and each consietin ~ of six sinq;le fibrils J were sub .coted to the same tree L.ment . To elL:iinote the possib111 ty of differences in \':eter c :m tent, lanolin fro,., the same container was used to ::1ske the mi x".;ures t."1.entioned used in bove . These mixtures wore then 11 the subsequent experiments . The increase in length of the material after three weeks is slown in teble 1 . TABLE 1. Lanoli n trealm enl s on cotton a1'.d hemp . . Cotton Treatm ent % total length increase in 21 days - H e mp ~ % total length increase Ave. % in 23 days -- No treatment ..... . . . ..... . • Lanolin o~Iy ............ . . . Lanolin - ~% indole acetic acid ... . .. ........ . .... . Lanolin - .2% acetic acid . . .38, .46, .14 .51 .31 .54, .50 0.00 0.00 .60, .66, .46, .60, .41 .55 .49 .60 0.19 0.18 .41 , 3. '11he indications are thi:,i t in v ar i ,)us mixt ures app lied to cotton thread , lanolin a lone is the · c2<u se of the tncrBt"Jsed extensib ility ,. where&s in he. 1p the increased e.xtens:i.b i l ity i s due to the org ~nic ecid i n the lano lin . II . Strips of different ma ter ia ls were r u , bed with l anol i n , 1 ~ .p lin-., 2 per c ent ( by wei ght) i ndo le ace tic aci d , l a n o i tn"':'. ;2. ;pe r c en t ~teetl c ittcld , and h m olin-,..2 per cent water . They we,re 7 . 5- l .5 c.m. Jong , de pending on the material . in any one e roup they were t he same l eng th, With- Each strip in tur n was fas tened in a horizontal posi tion on a wood en b lock \\ri th 2 . 5 c m. of i t .s be.se a bu tt i n g on e p:i_ece of wood . The r e - mainder of the str ip extended 'ho:r l zontaily an a. . f r ee in the ai r. A small lead rider was atta ched t o t he end of each piece , £md t h e ang le t l~ough wh i ch e a c h stri p b e n t f' ror.a t lie p l ane of t h e wood.en b loc k tvas J10 :2s u :rea b :i a protre.c tor . T 1e nieces were t hen l aid on a tab le, in diff use light , · at a temper ature of 23 c. After a period of time t he ang l e of b ending of r,.e: ch str ip wt1s ag ain meas ured . E.~cept in the cases of the c ardb oard strips , the f res h C he n ◊Dodium a l burr1 roots, and the i', nb ro s hi 2,silo s ta c hya stems a.nd roots , &11 the ma terial had been prevt ously dri ed i n an lu:~ r b arium pr ess . 'l'he . results ,_re pre sen ted in table 2,• .f.ffi inspec ti_o n of the se data. sho'lr-rn that t h ere are no c onstant , s pecific effects of any of the treatments for the mate rial used .. 4. TABLE 2. Lano lin treatments on stem and ro ot materials. A \'erage difference in degrees be tween original & fin a l cmTa lure (& stand. error) in: N umber of strips M aterial -·- - H ours of treatment - Lanolin --- Lan-indole acetic Lan-a cetic ac id L an-water - .2 ± 0 ± .8 ± - .4 ± 0 ± .2 ± .2 ± .4 .25 ± 1.3 1.0 ± .5 ---- S TEM: Ambrosia ...... . . .. . Pota to . . . .. . .. . . . . . Cardboard ....... . . . R oOT: Carrot ... .... . .. . . . W illow ... . . . . ... . . . C henopodi1tm . . . .. . Com Branch .. ... .. C om Brace ... . .. ... Ambrosia . . .. . . . ... Onion ..... ... ... ... Pistia &tratioles .... . Ltidwigia sp . ...... . Pea ... . . ....... . ... III . 5. 4 5 12 30 14 .2 ± 1.2 0± .6 .6± .12 5 4 1 34 30 17 20 18 13 29 32 32 20 7.6 ± 3.3 1.7 ± 1.1 3.0 - .6 ± .8 2.0 ± 3.3 4.5 ± 3.5 5.5 ± 3.0 24 .0 ± 7.7 2.0 ± .7 7.5 10 4 4 8 4 5 2 .7 .4 .09 3.6 ± 3.0 .1 .25 ± 1.0 -.1 ± .1 2.8 ± .8 2.0 ± 3.4 5.1 ± 2.2 24.0 ± 10.5 3.2 ± 1.4 13.0 .9 0 .05 3.4 ± 2.0 .25± .1 4.0 - .4 ± .6 2.8 ± .9 4.5 ±4.2 6.5 ±2.2 17.0 ± 9.0 3.8 ± 1.6 12.5 2.2 - 1.0 0 .3 2.5 .3 66 27 .0 7.0 14.0 ± 1.0 ± .7 ± .5 ± .7 ± 1.2 ± 3.2 ±1.1 ± 2.8 In vie, of' the r;rea t varia.b5.lity .found in the experiments carried out accordin:: to the iethods of Rob bins a.nd Jac kson ( 1937) , lt w._s decided to a0opt ot~er method s by which more consiste-:1t re sults , i ,ht ..., e obt ined . It was desirable to have (1) a . een s of controlling the i:mter content of t'•e ..uitf7rial used tmd (2) et''1.0d of me"surin g the e;,;:tenc:ib ilit.,t . material e. .. ore cc urate 1 c· c or..=11n , l y the rns sur jectec to exnerLent-1 conditions in v1 ter solu tioris , thus el in ting any differe nce8 in ".'B ter cont ent of the ori~inal m~te rial . The oxtensibtli ty wos :i"leasured nc - cura te l y to . 1 m~ . b• .e ans of a 1orizontal mic r oscope . The appurut.us consisted of a vortica.lly mounted ~lass 5. s ib l e to secure one end of t:l:e mate r ial under- investj_gation at the bottom by means of a ivire hook .. 'The other end vrn.s fastened to a .fine co pp6r wi re wh i c h r ~m up and over a pu l- ley ( hav:lng but lit tle frj.c t.ion ) and d own to a. p an fo r weigh ts . Supporting t he we\ght pan wa s a vertically movab le stage moun ted one. scr ew in order t o lower the weie;ht pan gr adual ly and thus !iiV o id a s udden a pp1 1catlon of tbe str e tc hing for•c e wh tch other wise mL,ht bre ak the m-r1te rial. At the top of the g l ass tube a r: :.t lli me t e r scale wa s mounted pe.rall e 1 to the c opper ·wire. Any c.•·,ang e in lenc; t h of the rrw.teri a l was re ad with the :alcrosc ope b y ob serving movement along t he sc a le of' an i ndicsitor· wh ich i.iva s pe r pe :·'.d:tcu1 arly e, t tnc hed to the wi re. 1~e b o ttom of the tuh e was equipped with a dr ain to fa cil it ate changing solut t on s • 'The rami 12: , c ot ton , and e.r ti fieisl si l k th.t'ends wor e secured wi tl.t sm.e ll l oops to t h e wire hooks at The roo t r:11:,te:riaJ. was ceme.n ted to wj_ r e t h e t op and b o ttom, loops h:v· means of h ard De Kho tln sky cement . T .. e E, e loops were then eas:i.ly p l aced on t h e WJ. r e hooks in t he tube , 'I'wo ... thread str and s of 1" i;;nnie ,, 35 cm. long , and Edng le str ands of No ., 3 c otton thr~ad t he se..me leng t h wex· e str e tche d by a 1 00 - gr ~mi weight ,;. 'Fhe sol uti ons u sed were vrnter, 0 . 00 1 I{ HCl and ~2 per cent i ndo le l;icetlc acid ( pH 3 " 3 ). For re.mie t h e re sul t s e.::q:n•e ssed as p crce nttlge to t al leng th increas e after 1 1 000 min utes 10:re: water , ., 75 ; . 001 W HCl, . 62 ; . 2 per 1 c ent :Lndole a eetic acid , •.60 . The r esul t s .for cotton &J:•E; g i ven for the fi r st 10- mi nut e per iod since on l y . 5 per c en t of the pe rcen tag e total incre ~se in leng th oc c u rr ed d ur ing 6. the next 99 hours. They were : witter, 2 . 78 per cent ; 0 ,. 001 N HC l, 2.06 per cent ; . 2 per cent indole acetic acid , l . 98 per cent; ,.2 per cent acetic e.cid , 2 . 0:3 per cent . Because of the slight extensibili t y of these ~aterials, more stati s ti cally si."'" nificant a ta r:erc not sought ,. From ther.e valu0s there is no indic ati c,.1 of. any specifi c increase of the extensibili ty due to p.H or the presence of' indole acetic acid . IV . Since ramie in pa.rticulor is known to hove nearly perfect orientation of the cellulose micelles parallel to the lon .... i tudinsl dil"ec tion of the fi ·~ er exi s , it was thou ..';, ht de- s irable to investig2te some ma terial with l~ss co plete :mi• cellar orientation . For thi s pur pose artificial si l k (re- generated cellulose) wa.13 used . . The strands consi s ted of two J threads, each of' whic L ·wcs composed of :.' ift:,r :.:·1hrils . They •. TABLE 3. Solution Extensibility of artificial silk in various solutions. Average percentage tota l length increase in 10 minutes with stand . error Water ... ....... ............. .. . 0.001 N H CI, pH 3.0 ........... . .2% Acetic acid, pH 3.3 .. ..... . .2% lndole acetic acid, pH 3.3 . . J % Trans Cinnamic acid . . . . . . .2% Oxalic acid .... .. .. . ..... . .2% Propionic aci d .. ......... . 13.5 ± .2 13.7 ± .2 16.l ± .5 16.2 ± .4 15.4 ± .1 15.6 ± .4 14.5 ± .3 the extensibi l ity of ,rtificial silk, •• t the effect is not spec 1fie , as shovm b·; tho feet thn t other or ganic a cids 7. act simi le.rly . 'rhi a phenomenon c~n not be explained on the ba sis that t h e org a11ic acids esterify the f r .e e b.yc roxy-1 group~ on the 2,3 , 6 ci,.r b on atoms o.f t h e g l ueose ring s of the cellu lose of a rt ific5.al silk. This i s shown by the following experiment: Stran ds of artificial silk were wea1tly acety l a ted by treating wi t h. acetlc anhydride~ Tl1eir ex tensibi lity was deter mined by s t re t c h ing them with a 100-grrun wei ght for 10 mi.nutes~ . It was found to be 15.6 per cent. or within the same ra:nae as • that for • 2 per cent acetic acid,. ~ Furthermove, allowing the artificial silk to soak ln water , 0 ~001 N HCl, .2 per cent acetic acid• or .2 per cent indole acetic acid for 21 hours and then stretc hing for 10 mi n utes g ave valu es similar to t 'lO Se ob tained above . TI:1ey were as fol lows (d a ta as percent .. a . e total leng th increase) : water , 13.4; 0 . 001 N HC l, 13 . 9; .2 per cen t indole acetic acid , 16.2; ~2 per cen t acetic acid., 16 .. 2 .. It may be concluded t1rnt certa tn non - growth ... pro1noting org en5.. c acids i.nc:rease t he ex tensibility of artificial s i lk over t hat in wa ter or in ,001 N HCl. The so lutions are ef- fec tive onl y dur:tng the time the stretching fo rce :'.Ls e.oting . 'Ibis is s lwwn by the ±'act t hat pretreatment with t he so l u t i ons i n q uesti on :ls withou t e ffect .. v. Roots., 30 cm. long , f rom an onion bulb g r owing in water e.nd f rom the wa t er p lant$ Pi s ~ str~tio_t e s and Ludw1gis. sp. were thoroughly dried in an her•b&.r 'i.um press . were less th&n 2 mm. in d lame ter. 'i111:e r oots ~they were stretehed (with 8. equal f orces) in water , in .2 per cent indole ac etid acid * and in .. 2 per cen t a,cetic acid., 'l1he results , in t he order n oted f or t h e sol ution s, were (as percen tag e t otal leng th q.10 , incre a se in 10 ~inutes): onion, 2 . 52/ 3 . 93; Pistia stratiote s, seen that t here is n o indt ca t ion tha t indo le a ce t t c acid h a.s caused a decreased exten sibility of root walls . Fresh vrnte1• cultured on i on roots were stretched in , the manner described in section II I . used . A wei ght of' 20. s gr arns was The roots were sel ected f0 r rm i f or ro.i ty of diameter and were about 13 cm. long .. S:lnce it was found that t he slope of t he e Ktens ibi l i t y~ t i me curve wos c onst an t after the fir s t 77 mi nut es , t , e reoding s presen ted were taken at that time. The results (e.. pre s sed as percen tage tota l lene,: th increase with standar d error) were : water, 3. 97 acetic ecid, 6 . 60 3 . 50 l. ">0 . • 09 ; • 2 per c ent ,. 68 ; . 2 >er cen t i ndole a cetic acid , F'rom :the s e da ta it is cle er t h nt t '.1e r oot mat- erial in ..2 per cen t :tndole acetic acid and i n t h e· . 2 p0r c ent ac et i c acid he.a a n incr eas ed ex ten sibi lity over tha t j_n water .. As t 1e val ues ob talned for t h e ac et i e a cid an d t h e i n- dole ac e tic acid do not show a s i 6 nificant sta t istical dif- . fe r ence , t h e effec t ob ser,red i s probab l y a ge nera l one fo r or- g an ic acids at pH 3 ,3. DISC JSSI DN. It was fo und dif'ficul t to dr aw d efinite cone l u s i ons reg arding cell- wall extensib il i ty b as e d on experimen ts in - s. volving dried stem and root materials with lano l ln a pplied to the ,1_. ':i.'b.e results of s u c h procedures showed va!r"iabili ty and inc on sistency . Thi s was probably due to ind iv.:i.due.1 di f .,. ferences in the ma terials . With an improved techn ique ; in one c ase ( that of artificial si l k ) staU.sticall,y EliGnif'::l.cant diff eren ces were obtained . If this b e accepted as an ex- a mple of stem eell wgJ. l ~-ie.terial , then . 2 per qent lndole acetic acid d ocs cause a s mall increa s e in the ex t ensib ility , but likewise so do other org an i c aci d s kn own n ot to b e growth- promoting substances in plan ts -. No in0i eetion of a decreased extensibility of root wall n1a tm... ie.1 as s ugge sted by Robb ins and t.T ackson ( 19 37) w~ts fmn1d . On t he contr r y , it wes ob served that ro ot ma t erial had an , increased extenslb:i.lity ln . 2 per cent indole ac e t i c acid as well as in . 2 per cent acetic acid . An 11 aci d effec , 11 has b e en noted by Bonner (1934) who found that Aven a col e op ti les at a pH of 4 .1 h a:ve a much grea t ~n· plast l city than t hose at e. pH of '7 , 1 ~ While the res u.lts r, iven in this paper Rre c oncerned with conc entrations ( . 2 per cent or 1,14 X 10 -J. M. ) of het ~ ero-- a uxin much hi gher than exist phy siolog ical l y , it i s inter .. er::itin g , never theless , to compare t hem with t h e e .ffects of the lower ones. Auxin at a physio l o '!: :tCH.l concent:rat t on he. s been s 1own by Heyn ( 1931 ~ 1934) t o increas e the p lastic exten si ... bi l i ty of' Avena c o leoptiles and Lupinus hypo cotyls . E.15dinc ( 1 933 ) h as found a siml l a r .ac t:L on of ph..:y:;iiological concentra ... ti ons of mudn on f lower stalks • Amlonf;~. (1937) has ob served 10. -<>t o 10- s M in- tha t a concentra t i ~n of hetero- eux in be twe en 10 creased t h e exten s i b ility of' roots of Vicia F'ab a. ., ti ons of Concentr a ... 6 16 to 10- 3 M c a used a decrease i n their extensib ility , - 2. wh ile a. c oncentr a ti on of 10 NI ·was f ◊tmd, i n. agreemen t wi t h t he work presen ted her e , to res ult i n an inc reased ex tensi b ility of non- 11vinJ roots . sm./l)1ARY ~f'he experiments of' Rot?b i ns ond J eeks on ( 193'7 ) concerning t he ext en sib :llity of stem an d root wa l ls were re peated i n as n e 8rly sn iden t ical manner as poss5.ble and found to g ive no c oncl usive evi dence a s to t he effe ct of gr owt h hor ... ,110:ne upon them. Artificial 811k was f ound to h ave an increrrned exten.. sib i li ty i n c ertain org an ic s.cids h.'TI. own n ot to be growt h s ub ... stances , a s well as in . 2 per cent i ndole ecetic acid. Oni on roots wer e .:Cound to have an increa s ed ex tensi b i li ty in a .2 per cent indole _acetic acid and in . 2 per cent acet i c acid . 11. Literature Cited .Am.long ,._ II . U. 1 93'7. Uber r'ie Eedeutu.ng d es Yiuchsstoffee fl't:r V-iachs tum und Ge ,) tropismus d er Viurzel. Ber. Deutsch. Bo t . Ge s. 55 : Bonner, J. 19;34. 21: 406-423 183•136 n1e r e la.tion of hydrogen ions to the Protoplasma erowth rate of the Av ena coleo pt ile. Heyn ., A . N . J. Rec 1931. Dor Mechanismus aer Zell n treokung _ Trev . Bot . Neerl. 28 : 113-244 H 'l ~A. :Die Plest:Lzitat d er ~::ellmemb ran unter Ein.f l ut,s v on Viuc h ss t of f'. Proc. K. Akad . \/f,tenschap . Amst er d a m 37: 180-132 Robbins , \Y . J, and J. R. J ackson. 1937 . Effect of 3i nd ole ace t ic acid on cell walls of st eru and r oot. Amer. J our. n ot. f :,:1: : 33.,. 37 1 933 . U'c er du s Wachstum cler Inflore sz enzscn© ft:e . J abrb. Wiss. f at . 77 : 627- 657 . .~'.od i n.e; , H . Went, F . V:! ., e.nd K. V . Th'im.snn . 1 937. New Y or k: . Phytohor mones.