A B S T R ACT I. STUDIES ON PLANT GROWTH SUBSTANCES A p a r t ic u la t e f r a c t io n from corn s e e d lin g s was found to con vert tryp top h an to in d o le a c e t ic a c id (IAA) in m inute amounts. None o f t h e c o - f a c t o r s examined appeared to in c r e a s e th e a c t i v i t y , and a c o n s id e r a b le p o r tio n o f th e a c t i v i t y was l o s t upon f r e e z in g . The a c t i v i t y sedim ented in th e s o - c a lle d m itochondral f r a c t io n s , but th e m icrosom al f r a c t io n a ls o con tain ed some a c t i v i t y . to s o lu b liz e th e a c t i v i t y were u n s u c c e s s fu l. A ll attem p ts When tryptophan-2-C 1^ was u sed a s s u b s tr a te , IA A -1-C ^ was i s o la t e d w ith no d ilu t io n o f s p e c ific a c t iv it y . In order to determ ine i f th e in v it r o system i s a c t u a lly o f s ig n i f i c a n c e , d ir e c t la b e lin g experim ents were attem p ted . I t was shown t h a t l i t t l e IAA i s p r e se n t in th e s e e d lin g s , alth ou gh th e f r e e tryptophan co n ten t was about 3 mg per kilogram . I t appeared d e s ir a b le , t h e r e fo r e , to d evelop an is o l a t i o n method which would be s u it a b le w ith la r g e r amounts o f corn s e e d lin g t i s s u e . T h is work made i t apparent th a t n ot more than th e order o f 10 o f IAA. was jig p r e se n t per kilogram o f r a p id ly growing e t i o la t e d corn s e e d lin g s . A second phase o f experim ents in th e realm o f p la n t au xin s con­ cern s s t u d ie s o f th e e f f e c t o f N -l-n ap h th ylp h th alam ic a c id on th e growth and th e g eo tr o p ic resp on se o f s e e d lin g s , and a stu d y o f th e a c t i v i t y o f t e t r a z o l e an alogu es o f IAA and 2 ,4 -d ic h lo r o p h e n o x y a c e tic a c id ( 2 ,4 - D ) . N -l-n ap h th ylp h th alam ic a c id (NPA) was shown to in h ib it th e g e o tr o p ic resp o n se o f s e e d lin g s d is p r o p o r tio n a lly as compared to growth. S tu d ie s on th e t e t r a z o l e s dem onstrated th a t a 5 ' - t e t r a z o l e rin g may r e p la c e th e carb oxyl group o f IAA, w ith th e r e t e n tio n o f growth prom oting a c t i v i t y in th e Avena s e c tio n t e s t . The 2 ,4 -D an alogu e, on th e o th er hand, was an in h ib it o r o f endogenous growth, IAA induced growth, and 2,^ -D induced growth. Though th e in h ib it io n o f IAA in ­ duced growth was n o n -c o m p e titiv e , th e in h ib it io n o f 2,^-D induced growth in d ic a te d a stro n g c o m p e titiv e component. T his le d to th e c o n c lu sio n th a t IAA and 2 ,4 -D a c t a t d if f e r e n t growth ind u cin g s i t e s , or th a t th e r e was co m p etitio n a t a tr a n sp o r t or uptake s i t e . Addi­ t i o n a l work on in t a c t bean p la n ts in d ic a te d th a t an uptake s i t e may be in v o lv e d . II, ISOLATION AND PARTIAL CHARACTERIZATION OF THE PRECURSOR OF 6-METH0XYBENZ0XAZ0LIN0NE IN ZEA MAYS The work on a n a tu r a lly occu rrin g sw eet compound in corn seed ­ l i n g s was th e r e s u lt o f th e d i f f i c u l t y encountered in se p a r a tio n o f t h i s compound from IAA in e x tr a c ts o f corn s e e d lin g s . A compound (C ^ c ^ N ) was i s o la t e d in c r y s t a lin e form, and found to c o n s t it u t e about 0 .5 $ o f th e dry w eig h t o f th e s e e d lin g s . I t appeared to be i d e n t i c a l to th a t is o la t e d by R. J. Suhadolnik (Ph.D. T h e s is, P en n syl­ van ia S t a te U n iv e r s it y . 1 9 5 6 ), who proposed th e s tr u c tu r e 2 ,5 - d i h yd ro x y -3 ,4 -m eth len ed io x y p h en ylacetam id e. P resen t s tu d ie s in d ic a te d th a t th e proposed s tr u c tu r e was in c o r r e c t , and f i n a l l y th a t th e com­ pound was r e a d ily converted to 6-m ethoxybenzoxazolinone w ith a l o s s o f form ic a c id , as r e c e n t ly rep orted by Wahlroos and V irtan en (Suomen K e m is t ile h t i B 3 2 sl39-1^ 0, 1 9 5 9 ). These au th ors su g g e st th e s tr u c tu r e l,3 -d ih y d r o x y -2 -k e to -6 -m e th o x y -l,4 -b e n z o x a z in e , and our data appear to f i t t h i s s tr u c tu r e . ^ r I. STUDIES ON PLANT GROWTH SUBSTANCES (and) II. ISOLATION AND PARTIAL CHARACTERIZATION OF THE PRECURSOR OF 6-METHOXYBENZOXAZOLINONE in zea mays By Robert H ille r y H am ilton, J r. A THESIS Subm itted t o th e S chool fo r Advanced Graduate S tu d ie s o f M ichigan S t a te U n iv e r s ity in p a r t ia l f u lf i llm e n t o f th e requirem ents fo r th e degree o f DOCTOR OF PHILOSOPHY Department o f Botany and P la n t P ath ology I960 ProQuest Number: 10008617 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10008617 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346 I should l i k e to exp ress my a p p r e c ia tio n t o P r o fe sso r R. S. Bandurski fo r h is many su g g e stio n s and a s s is t a n c e during th e s e s t u d ie s . I would a ls o l i k e to thank Dr, Te May Ching and Dr. A leks K iv ila a n who c o n tr ib u te d so much to th e s tu d ie s on s y n th e tic p la n t growth su b sta n ce s. I t i s a ls o a p le a su r e to acknowledge th e many s u g g e stio n s o f D rs. L. G. W ilson, W. H. Reusch and P r o fe sso r R. M. H erbst. T A B LE OF CON T E N T S INTRODUCTION I. II. S tu d ie s on P la n t Growth Substances A. Occurrence and B io s y n th e s is o f In d o le -3 -A c e tic A c i d ........................................................................ . . . . . . . o Page 2 B. S y n th e tic P la n t Growth Substances . . . . . . . . . Page 3 I s o l a t io n and P a r t ia l C h a r a c te r iz a tio n o f th e P recursor o f 6 -M ethoxybenzoxazolinone in Zea mays . . . . . . . . Page 4 REVIEW OF LITERATURE I. S tu d ie s on P la n t Growth Substances A. O ccurrence and B io s y n th e s is o f I n d o le -3 -A c e tic A cid 1. 2. 3. 4. 5. 6. 7. B. Work on th e E x iste n c e and Nature o f P lan t Hormones . . . . . . . . . . . . . . . . . . . . D e te c tio n o f I n d o le -3 -A c e tic A cid in Higher P la n ts . . . . . . . . . . . . . . . . . . . . . The E x iste n c e o f I n d o le -3 -A c e tic A cid in Avena C o le o p t ile s . . . . . . . . . . . . . . . . . . . Lack o f R e d is tr ib u tio n o f Exogenous I n d o le -3 A c e tic A cid F ollow in g Geo- or P h o to tro p ic S tim u la tio n . . . . . . . . . . . . . . . . . . . Bound or Conjugated Forms o f IAA . . . . . . . . Other In d o le Compounds Occurring in P la n ts . . . B io s y n th e s is o f I n d o le -3 -A c e tic A cid . . . . . . S y n th e tic P la n t Growth Substances Page 5 Page 8 Page 13 Page 15 Page 16 Page 18 Page 20 . . . . . . . . . Page 25 I I • I s o l a t io n and P a r t ia l C h a r a c te r iz a tio n o f th e P recursor o f 6-M ethoxybenzoxazolinone in Zea mays . . . . . . . . Page 84 T A B L E OF C 0 N T E N T S (Continued) EXPERIMENTAL I. S tu d ie s on P la n t Growth Substances A. Occurrence and B io s y n th e s is o f I n d o le -3 -A c e tic A cid . !• 2. 3. B, . Page 28 . Page 44 . Page 51 . Page 75 The E ffe c t o f N -l-N aph tylphth alm ic A cid on th e Growth and G eotropic Response o f S e e d lin g s (See R ep rin t) . . . . . . . . . . . . . . . . . B io lo g ic a l A c t iv it y o f T etr a z o le Analogues o f I n d o le -3 -A c e tic A cid and 2 S4-D. (See R ep rin t) Page 79 S y n th e tic P la n t Growth Substances 1. 2. II. In V itro Experim ents on the B io s y n th e s is o f I n d o le -3 -A c e tic A cid from Tryptophan . . . . . S tu d ie s on th e I s o la t io n o f I n d o le -3 -A c e tic A cid . a . I s o la t io n o f 3 - I n d o ly la c e t ic A cid from U s tila g o zeae Tumors . . . . . . . . . . . b. S tu d ie s on I s o la t io n o f I n d o le -3 -A c e tic Acid from Corn K ernels and E tio la te d Corn S e e d lin g s . . . . . . . . . . . . . . . . In Vivo L a b elin g Experim ents on th e B io­ s y n th e s is o f I n d o le -3 -A c e tic A cid. . . . . . . I s o l a t io n and P a r t ia l C h a ra cte r iz a tio n o f th e P recu rsor o f o-M ethoxybenzoxazolinone in Zea mays. . . . . . . . . LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . Page 81 Page 84 Page 108 I NT RODUCT I ON The hormonal c o n tr o l o f p la n t growth has been in v e s t ig a t e d by a la r g e number o f p la n t p h y s i o lo g i s t s , p a r t ic u la r ly s in c e th e d isc o v e r y by F. W. Went o f a q u a n tit a tiv e experim ental approach. In s p it e o f th e a rra y o f in t e r e s t in g p h y s io lo g ic a l exp erim en ts, much chem ical and b io ch em ica l work rem ains. I t may b e, however, th a t th e p r e se n t s t a t e o f chem ical tech n iq u e i s not adequate t o d e a l w ith problems on th e hor­ monal l e v e l . Q u estion s such as th e fo llo w in g a re n ot p r e s e n tly ea sy to d e a l w ith e x p er im e n ta lly . What are th e hormones o p era tin g in th e v a r io u s t i s s u e s o f a p la n t a t v a rio u s s ta g e s o f i t s l i f e c y cle? hormones, l i k e th e v ita m in s, to be regarded as co-enzym es? Are a l l I f n ot co­ enzym es, how may th e hormones i n i t i a t e th e ch ain o f r e a c tio n s th e y do in d eed tr ig g e r ? How can one be c e r t a in th a t compounds is o la t e d from p la n t e x tr a c ts a c t as hormones in v iv o ? Why do d iv e r s e s y n th e tic com­ pounds have s im ila r b io lo g ic a l a c t i v i t y , a t l e a s t in a q u a l it a t iv e manner? The ex p erim en tal work to be p resen ted w i l l be in th e form o f f i v e papers p lu s one a d d itio n a l p o r tio n on th e b io s y n th e s is o f 3 - in d o le a c e t ic a c id (IAA). The l a t t e r p o r tio n , and two papers concern ing i s o l a t i o n o f IAA from mature corn s t a l k s , tumors and corn s e e d lin g s , c o n s t it u t e P art A o f S e c tio n I on a u x in s. P art B o f S e c tio n I w i l l concern s y n th e tic a u x in s, to be p re sen te d in th e form o f two p u b lish ed papers which d e a l w ith th e e f f e c t o f N -l-n a p h th ylp h th alam ic a c id on growth and th e geo­ tr o p ic resp o n se in s e e d lin g s , and th e b io lo g ic a l a c t i v i t y o f t e t r a z o le a n a lo g u es. S e c tio n I I i s concerned w ith i s o l a t i o n and c h a r a c te r iz a tio n o f a sw eet p h e n o l-lik e compound from corn s e e d lin g s . A g e n e ra l review on au xin s i s p resen ted f i r s t because each paper co n ta in s a ra th er s p e c i f i c and lim it e d rev iew . -2 - I. STUDIES ON PLANT GROWTH SUBSTANCES A. Occurrence and B io s y n th e s is o f I n d o le a c e tic A cid In th e p r e s e n t work an e f f o r t was made to avoid th e c r u c ia l, but n eb u lo u s, q u e stio n s enumerated in th e in tr o d u c tio n . f i c u l t i e s were encountered. N o n e th e le ss, d i f ­ I t was in ten d ed to in v e s t ig a t e a w e ll- d e fin e d problem o f r e s t r i c t e d scope (th e b io s y n t h e s is o f in d o le -3 a c e t i c a c id from tr y p to p h a n ), but soon q u e stio n s a ro se about th e assum ptions on which th e problem was based. F i r s t , i t would be d e s ir ­ a b le t o prove th a t in d o le - 3 - a c e t ic a c id (IAA) was, in f a c t , sn y th e s iz e d from tryptophan in v iv o . S econ d ly, i t i s apparent th a t one must be a b le t o measure i n i t i a l and f i n a l IAA co n cen tr a tio n in th e s e e d lin g s i f one i s to determ ine s p e c i f i c a c t i v i t i e s o f IAA sy n th e s iz e d from tryp top h an . A p a r t ic u la t e f r a c t io n from corn s e e d lin g s was found to con vert tryptophan t o IAA in m inute amounts. None o f th e c o - fa c t o r s examined appeared t o in c r e a s e th e a c t i v i t y , and a c o n sid e r a b le p o r tio n o f th e a c t i v i t y was l o s t upon f r e e z in g . The a c t i v i t y sedim ented in th e s o - c a lle d m itochondral f r a c t io n , but th e microsomal f r a c t io n a ls o con­ ta in e d some a c t i v i t y . s u c c e s s f u l. A ll attem p ts to s o lu b liz e th e a c t i v i t y were un- When tryptophan-2-C 14 was used as s u b s tr a te , IAA-l-C n Zi was i s o l a t e d w ith no d i lu t io n o f s p e c if ic a c t i v i t y . In order to determ ine i f th e in v it r o system i s a c t u a lly o f s i g n i ­ f ic a n c e , d ir e c t la b e lin g experim ents appeared to be e s s e n t i a l . Other work in t h i s la b o r a to r y in d ic a te d l i t t l e r a d io a c t iv it y could be o b ta in ­ ed in IAA and tryptophan by fe e d in g w ith in d o le . C ^ C >2 to e t io la t e d corn s e e d lin g s In th e s e exp erim en ts, about 25 grams o f s e e d lin g s -3 14 were fe d about 1 me o f C Ogi and th e i s o l a t i o n o f IAA and tryptophan f a c i l i t a t e d through a d d itio n o f c a r r ie r n o n -la b e le d IAA and tryptop han . In th e p r e se n t work i t was a ls o shown th a t l i t t l e IAA i s p r e se n t in th e s e e d lin g s , alth ou gh th e f r e e tryptophan co n ten t was about 3 mg p er k ilogram . I t appeared d e s ir a b le , t h e r e fo r e , to d evelop an i s o l a ­ t i o n method which would be s u it a b le w ith la r g e r amounts o f corn s e e d lin g tis s u e . T his made i t apparent th a t not more than th e order o f 10 jig o f IAA was p r e se n t per kilogram o f r a p id ly growing e t i o la t e d corn s e e d lin g s . B. S y n th e tic P la n t Growth Substances A second phase o f experim ents in th e realm o f p la n t au xin s concerns s tu d ie s o f th e e f f e c t o f N -l-n ap h th ylp h th alam ic a c id on th e growth and th e g e o tr o p ic resp o n se o f s e e d lin g s , and a stu d y o f th e a c t i v i t y o f t e t r a z o le an alogu es o f IAA and 2 ,4 -d ic h lo r o p h e n o x y a c e tic a c id (2 ,4 - D ). N - l- naphthylphthalam ic a c id (NPA) was shown to in h i b it th e g eo tr o p ic resp on se o f s e e d lin g s d is p r o p o r tio n a lly as comparted to growth. This appeared to c o n f l i c t w ith th e c l a s s i c a l th e o r y o f Went and Cholodny, or to in d ic a t e th a t l a t e r a l p o la r it y or r e d is t r ib u t io n o f auxin was p rev en ted . S tu d ie s on th e t e t r a z o l e s dem onstrated th a t a 5 ' - t e t r a z o l e r in g may r e p la c e th e carb oxyl group o f IAA, w ith th e r e te n tio n o f growth prom oting a c t i v i t y in th e Avena s e c t io n t e s t . The 2 ,4 -D an alogu e, however, was an in h ib it o r o f endogenous growth, IAA induced growth, and 2 ,4 -D induced growth. Though th e in h ib it io n o f IAA induced growth was n o n -c o m p e titiv e , th e in h ib it io n o f 2 ,4 -D induced growth in d ic a te d a stron g c o m p e titiv e component. T his le d t o th e in t e r e s t in g c o n c lu sio n th a t IAA and 2 ,4 -D a c t a t d if f e r e n t growth in d u cin g s i t e s , or th a t th e r e was c o m p etitio n a t a tr a n sp o r t or uptake s i t e . A d d itio n a l work on in t a c t bean p la n ts in d ic a te d th a t an -4 uptake s i t e may be in v o lv e d . II. ISOLATION AND PARTIAL CHARACTERIZATION OF THE PRECURSOR OF 6 -METHOXYBENZOXAZOLINONE IN ZEA MAYS The work on a n a t u r a lly o ccu rrin g sw eet compound in corn seed ­ l i n g s was th e r e s u lt o f th e d i f f i c u l t y encountered in se p a r a tio n o f t h i s compound from IAA in e x t r a c ts o f corn s e e d lin g s . A compound (C^H^O^N) was i s o l a t e d in c r y s t a lin e form , and found to c o n s t it u t e about 0 .5 $ o f th e dry w eig h t o f th e s e e d lin g s . I t appeared to be i d e n t i c a l to th a t i s o l a t e d by R. J. Suhadolnik (1 4 8 ), who proposed t h e s tr u c tu r e 2 , 5 - d ih y d ro x y -3»^ -m ethylened ioxyph en ylacetam id e. Our s tu d ie s in d ic a te d th a t th e proposed s tr u c tu r e was in c o r r e c t , and f i n a l l y th a t th e compound was r e a d ily converted t o 6-m ethoxybenzoxaz o lo n in e w ith a l o s s o f form ic a c id , as r e c e n t ly rep o rted by Wahlroos and V irtan en (173)* These au th ors su g g e st th e s tr u c tu r e 1 , 3-d ih yd roxy- 2 -k e to -6 -m e th o x y -l,il'»benzoxazine, and our data appear to f i t t h i s s tr u c ­ tu r e . -5 - I. STUDIES ON PLANT GROWTH SUBSTANCES A. Occurrence and B io s y n th e s is o f I n d o le -3 -A c e tic A cid in P la n ts 1. Work on th e E x is te n c e and Nature o f P la n t Hormones R e a liz a tio n th a t a lo c a liz e d area a t th e t i p s o f sh o o ts and r o o ts was concerned w ith g eo tr o p ic and p h o to tro p ic r e sp o n se s, may be a t t r i ­ buted t o Darwin (3 ^ ). He observed th a t th e c o l e o p t il e o f canary g r a s s, P h a la r is c a u a r ie n s is , was p a r t ic u la r ly s e n s i t iv e t o l i g h t and g r a v ity . Even though th e reg io n o f bending was below th e t i p , removal o f th e t i p p rev en ted th e resp o n se. The s e e d lin g would not respond to l i g h t even i f th e t i p was m erely covered p r io r to u n ila t e r a l illu m in a tio n , Boysen- Jensen (2 ^ ), working w ith o a t c o le o p t ile s , was a b le to show th a t c o n tin ­ u i t y o f l i v i n g protoplasm was not e s s e n t i a l fo r tr a n sm issio n o f th e s tim u lu s . A tr a n s v e r s e cu t d id not a f f e c t th e cu rvatu re resp on se and, in f a c t , replacem ent o f an e x c is e d t i p allow ed th e cu rvatu res to d ev elo p . P a a l (117) dem onstrated th a t when th e e x c is e d t i p was rep la ced w ith o n ly one s id e o f th e stump co v ered , th e covered s id e grew f a s t e r ; r e s u lt in g in a cu rvatu re n ot u n lik e normal geo- or p h o to tro p ic cu rv a tu re. The stim u lu s could p a ss through a la y e r o f g e l a t in , but not mica or platinum fo il. P aal was f i r s t to su g g e st a hormone was r e s p o n s ib le fo r geo- and p h o to tr o p ism s, w h ile Sdding (1^0) showed th a t replacem ent o f th e e x c is e d t i p r e s to r e d most o f th e growth l o s t by d e c a p ita tio n . F it t in g (^-l) was th e f i r s t in v e s t ig a to r to ap p ly th e term hormone in p la n t p h y sio lo g y . He a p p lie d th e term to a su b stan ce e x tr a c te d from th e p o l l i n i a o f t r o p ic a l o rch id s which induced s w e llin g of th e ovary when -6 a p p lie d to th e stig m a . About tw enty years l a t e r , Laibach ( 8 7 ) found th e s e e x t r a c ts c o n ta in ed a u x in , and orch id o v a r ie s responded to h etero a u x in a p p lic a t io n s , H aberlandt (57) observed i s o la t e d p o ta to tu b er parenchyma did not d iv id e u n le s s a sm a ll b i t o f phloem t i s s u e was p r e s e n t, and th a t th e phloem cou ld s t i l l induce d iv is io n when sep arated from th e parenchyma by a g a r. Crushed c e l l s were a ls o e f f e c t i v e in in d u cin g th e resp o n se. Haberlandt concluded two hormones were req u ired fo r th e resp on se; one a wound hormone coming from th e cu t s u r fa c e , and th e o th er a phloem hormone. The c l a s s i c a l s tu d ie s o f F. W. Went (177»178) r e s u lt e d in developm ent o f a q u a n t it a t iv e b io a ss a y fo r th e growth hormone o f Avena c o l e o p t i l e t i p s . . Went found t h a t i f th e e x c is e d c o l e o p t il e t i p was p la ced on a v ery sm all b lo ck o f a g a r, some o f th e a c t iv e su b stan ce cou ld be c o lle c t e d in th e agar. I f th e b lo ck was p la ced a s y m e tr ic a lly on a d e c a p ita te d stump, a cu rvatu re d ev elo p ed . Under a s p e c if i e d s e t o f c o n d itio n s , th e cu rvatu re was found to be d i r e c t l y p r o p o r tio n a l t o c o n cen tra tio n (number o f t i p s allow ed to d i f f u s e , or tim e o f d if f u s io n ) over a lim it e d range. This a ssa y made p o s s ib le chemi­ c a l work on th e n atu re o f th e a c t iv e su b sta n ce, and a ls o p erm itted many in t e r e s t in g p h y s io lo g ic a l exp erim en ts. Went was fu r th e r a b le to measure th e r a t e o f d if f u s io n o f th e su b stan ce through agar and th u s t o e stim a te th e m o lecu la r w eig h t to be about 376. b o ilin g and l i g h t . The a c t iv e su b stan ce was s ta b le to Thus th e i s o l a t i o n and chem ical s tr u c tu r e o f th e a c t iv e su b sta n ce became an a t t r a c t iv e problem , and i t was i n t e n s iv e ly in v e s t ig a t e d , e s p e c i a l l y by Kdgl, Haagen-Sm it, and co-w orkers ( 7 9 » 8 0 ,8 1 ,8 2 , 8 3 ) . N ie ls e n (112) had a lr e a d y found in 1928 th a t th e c u ltu r e medium from Rhizopus su in u s was r ic h in su b stan ce ca u sin g Avena c u r v a tu r e s. He l a t e r found ( 1 1 3 ) th a t th e su b sta n ce was e th e r s o lu b le but r e a d ily in a c tiv a t e d by p e r o x id es norm ally p r e s e n t. Furtherm ore, N ie ls o n a ch iev ed a p a r t ia l -7 p u r if ic a t io n such th a t 0 ,0 2 fug gave a cu rv a tu re. Dolk and Thimann (36) were a b le to determ ine a pK o f about 4 .7 5 fo r th e a c t iv e su b stan ce from Rhizopus c u ltu r e f i l t r a t e by shaking w ith e th er a t v a r io u s b u ffered pH v a lu e s . The su b stan ce seemed to be s e n s i t iv e to o x id a tio n and warm a c id s , bu t s ta b le to warm a l k a l i . At about th e same tim e, Kttgl and Haagen-Smit rep o rted p relim in a ry p u r if ic a t io n o f a c id au xin s from Rhizopus r e f le x u s , y e a s t , and E. c o l i (8 0 ). Kdgl, Haagen-Smit and Erxleben (82) a ls o found human u rin e to be a v e ry r ic h source o f a c t iv e m a te r ia l and is o la t e d 40 mg o f a c t iv e c r y s t a ls from about 33 g a llo n s . This rep resen ted about a 50*000 f o ld p u r if ic a t io n as measured by th e Avena cu rv a tu re t e s t . The formula fo r th e a c t iv e su b sta n ce , c a lle d auxin a , was C^sH^O^ and th e s tr u c tu r e appeared to be 2 , 4 - d i i s o b u t y l - l ^ -c y c lo p e n te n e y l-o t,^ , £ -tr ih y d r o x y v a le r ic a c id . L ater th e y i s o la t e d from corn o i l and b a r le y m alt a second a c t iv e su b stan ce c a lle d auxin b (7 9 )o f th e Auxin b could be con verted to auxin a by d eh ydration hydroxyl group, g iv in g r i s e to th e J3 k eto d e r iv a t iv e ( 7 9 ). S t i l l another compound (81) was p r e se n t in th e u rin e c o lle c t e d a t a h o s p it a l from a p a r tic u la r 1 8 -y e a r -o ld you th . This is o la t e d compound was c a lle d h e ter o a u x in , and i t was found to be id e n t ic a l t o in d o le - 3 - a c e t ic a c id , o r i g i n a l l y i d e n t i f i e d from u rin e by Saikow ski (129) , and sy n th e s iz e d by E llin g e r (3 ? )Thimann (157)* in l a t e r work on th e a c t iv e m a te r ia l from Rhizopus s p . , was a b le to o b ta in a minute amount o f c r y s t a lin e m a te r ia l by vacuum su b lim a tio n which appeared id e n t ic a l in a l l r e s p e c ts to in d o le - 3 - a c e t ic ac id ( IAA) . Auxins a and b have not been su b seq u en tly i s o la t e d . W ieland, e t a l . (181) have r e c e n t ly fo llo w e d th e method o f K 8gl, Haagen-Sm it, and E rxleben _8(8 2 ) f o r th e i s o l a t i o n o f auxin a from u rin e and ob tain ed o n ly th e m ethyl e s t e r o f in d o le - 3 - a c e t ic a c id (IAA). This was an a r t i f a c t produced by b o i l i n g in m eth an olic HC1 w hich was th o u g h tto la c t o n iz e auxin a . Brown, H enbest, and Jones (26) have prepared l , 5 “Cyclopentenyl*^3-keto-,£h y d r o x y v a le r ic -X -la c to n e . This c lo s e analogue o f auxin b appears to e x i s t o n ly as th e la c to n e in aqueous s o lu t io n w ith t h e ^ - k e t o group r e a d ily e n o liz in g . Other d if f e r e n c e s were a ls o found between t h is d e r iv a t iv e and auxin b la c to n e . The accum ulating evid en ce has s lo w ly le d to th e o p in io n th a t auxin a and auxin b do not in f a c t e x i s t . The e a r ly work, however, seemed to in d ic a te th a t auxin a was probably th e auxin o f h ig h er p la n t s . These c o n c lu sio n s were based on d eterm in ation o f th e m olecu lar w eigh t by th e d if f u s io n tech n iq u e, and th e p a r t ia l a c id s t a b i l i t y . Larsen (92) has review ed th e m ethodology and r e s u lt s o f v a rio u s i n v e s t i ­ g a tio n s u sin g th e d if f u s io n tech n iq u e to e stim a te th e approxim ate m olecular w eig h t o f au xin e x tr a c te d or d if f u s in g from h igh er p la n t t i s s u e s . There a re many d i f f i c u l t i e s , n o t th e l e a s t o f which i s th e p resen ce o f f r e e l y d i f f u s i b l e in h ib it o r s an d /or more than one a c t iv e su b sta n ce. Most recen t d eterm in a tio n s have r e s u lte d in v a lu e s more c l o s e l y corresponding to IAA than t o au xin a or b . T erp stra (153) has p o in ted out th a t a t t r ib u t in g th e a c id s t a b le p o r tio n o f th e a c t i v i t y to auxin a may be in erro r. She found t h a t th e r e s id u a l a l k a l i and a c id s ta b le a c t i v i t y corresponded, a f t e r paper chromatography, to an Rf o f IAA. 2. D e te c tio n o f I n d o le -3-A c e tic A cid in Higher P la n ts The ca se o f IAA as a n a tu r a l p la n t auxin was much b o ls te r e d by th e i s o l a t i o n o f IAA a f t e r a lk a lin e h y d r o ly sis o f corn seed (1 7 ,5 5 ) and th e i s o l a t i o n o f "free" IAA from immature com seed ( 5 4 ). The p h y s io lo g ic a l s ig n if ic a n c e o f th e v ery h igh co n c e n tr a tio n o f IAA in corn seed i s unknown. -9 H o lle y e t a l . (6?) sep a ra ted th e a u xin s o f cabbage by means o f cou n ter cu r re n t d is t r ib u t io n u sin g d ie t h y l eth er and pH 6 .0 phosphate b u ffe r . They found two a c t iv e compounds, one o f which had th e same d is t r ib u t io n c o e f f i c i e n t as IAA, and another much more w a te r -s o lu b le . S in c e th e development o f paper chrom atographic tech n iq u e, many r e p o r ts o f d e t e c tio n o f IAA (and oth er growth prom oting and in h ib it in g su b sta n ce s) may be c i t e d . The com piled l i s t (Table I ) makes i t apparent th a t IAA and o th er in d o le compounds have been d e te c te d in a number o f p la n t s . C aution shou ld be employed w ith regard to rep o r ts in which IAA i s re p o rted on th e b a s is o f b i o l o g i c a l a c t i v i t y a t th e proper Rf a f t e r u n id ir e c tio n a l chromatography in one s o lv e n t . Salkow ski and E h rlich r e a g e n ts are n o t, however, s e n s i t iv e enough to a llo w d e te c tio n o f IAA a t c o n c e n tr a tio n s d e te c ta b le in th e v a rio u s b io a s s a y s . The most popular b io a s s a y has been th e Avena s e c tio n t e s t ( 1 3 ). though Avena cu rvatu re (1 8 9 ), C ress root in h ib it io n ( 1 0 8 ), pea ro o t in h ib it io n ( 1 0 ), wheat s e c t io n s ( 9 8 ) , Avena f i r s t in tern o d e (115)» and in h ib it io n o f Coleus a b c is s io n (99) are some o f th e b io a ssa y tech n iq u es u sed . I t appears th a t th e new spray reagen t p-dim ethylam inocinam aldehyde proposed by H artley-M ason and Archer (59) w i l l fin d w ide u s e . T his aldehyde g iv e s c o lo r s w ith many in d o le compounds and i s about 10 tim es as s e n s i t i v e as p-dim ethylam inobenzaldehyde (E h r lic h 1s r e a g e n t). The s p e c i f i c i t y and r e l i a b i l i t y o f c o lo r rea g en ts fo r IAA le a v e s much to be d e s ir e d , e s p e c i a ll y from th e q u a n tit a tiv e sta n d p o in t. Even a s id e from t h i s , E h rlich and Salkow ski rea g en ts g iv e c o lo r s w ith a number o f p h en o lic su b sta n c e s (1 4 2 ,1 4 6 ). A lso , Salkow ski rea g en t i s n ot r e l i a b l e in th e p re se n c e o f red u cin g su b sta n ces (1 2 0 ). Both E h rlich and Salkow ski re a g e n ts were found to g iv e c o lo r s a t th e Rf o f IAA on paper chromatograms -1 0 TABLE I R eports o f O ccurrence o f I n d o le -3 -A c e tic A cid and R elated In d o le Compounds in Various Higher P la n t T issu e s P la n t T issu e & R eferen ces Compounds Found Type o f E xtract B ioassay B lack c u r r e n t-s e e d s , f r u it s ^ ® G o o seb er r y -se ed s, f r u i t s A pple seeds^-®° IAA ,IAN IAA IAA Acid Ether Wheat Sect.& it it Coleus n n a b c is s io n Avena c o l e o p t il e tip s ^ -^ IAA D iffu s a te Avena c o l e o p t i l e t i p s 153 IAA Color D etection Yes ? ? Oat Curvature No j ET2 O & H2 O Oat Curvature No Corn seed (im m ature)-^ IAA I s o la t io n Corn seed^-5 IAA H yd rolysis ( is o la t e d ) Corn s e e d ^ IAA Corn seed ^ -5 IAA, +Unk. A q .aceton e Corn s e e d ^ IAA Ether Whe<-t S e c t. Yes Corn s e e d ^ ^ IAA Ethanol Oat S e c t. Yes Corn s e e d ^ 9 IAA,IAAld Ether Oat Curvature Yes Corn p o l l e n ^ IAA Ether Oat S e c t. Yes 30 Pea seed & se e d lin g s ^ IAA,I Car. Ether Wheat S e c t. Yes Grape-^-5 IAA Ether Oat S e c t . , Oat Internode No 115 it - - it Yes IAA ti ti Ether tt n Oat Intern od e it 11 it 11 No it 11 IAA IAA,2 unk. Ether it Oat S e c t . it ti Yes it IAA Ether Oat Curvature Yes 98 B r o c c o li le a v e s-7 IAA Ether Wheat S e c t. No B r o c c o li le a v e s 9 6 IAA Ethanol Oat (p a s te ) Yes Asparagus f r u i t ^ IAA Ether Wheat S e c t . No S tra w b erry ^ ^ IAA Ether Oat S e c t. No P .t r ic u s p id a t a crown g a l l Tomato o v a ry H 5 Immature bean seed-^-^-5 C itr u s flow ers?"^ Corn endosperm Datura o v u le tumors 128 - 11TABLE I (con tin u ed ) P la n t T issu e P o ta to p e e l & Type o f E xtract Compounds Found R eferen ces 19 B io a ssa y Color D e te c tio n IBA Ether Oat S e c t. No Cabbage^? »39 IAA,IAN Ethanol Oat (p a s te ) Yes Cabbage*^ I . Car. E thanol Yes Cabbage” ' IAld E thanol Yes C abbage^ IAN ( I s o la t e d & i d e n t if i e d ) S k a to le Ether C abbage^ IAA Ether Oat S e c t . Sunflow er tumor Corn endosperm ^ IAA 7 7 7 ? Cabbage 97 Tomato crown g a l l tumor Normal tomato stemr tt 32 Yes Yes tt IAA, I .C a r , I . Car. Ether Ether Hevea le a v es* ^ IAA Ether Oat S e c t . No mK 101,102 Tobacco ovarys IAA,IAN Ether Oat Curvature No Ethanol Oat S e c t , Pea-Tomato Yes Ether Oat S e c t . No Ethanol Oat S e c t. No T o b a c c o ^ ^ . U n k .ind ole Yes Yes T o b a c c o * ^ IAA Sunflow er shoots-*-3 Bean & pea r o o ts & shoots-*-3 Corn r o o ts IAA & unks P o ta to p e e l? ? P o ta to sh o o ts 77 IAA, IAN IAA Ethanol P o ta to t u b e r s " ^ IAA H y d ro ly sis Cacao & bananas^"** IAA Ether Wheat S e c t. No Wheat roots^-5 IAA Ether Oat S e c t. No Pea s h o o t s ^ IAAld Ethanol Oat S e c t. Yes I . Car. IAld IAN IAAld IBA = = = = = in d o le - 3 - c a r b o x y lic in d o le -3 -a ld e h y d e i n d o l e - 3 - a c e t o n it r i l e in d o le a c e ta ld e h y d e in d o le b u te r y ic it it it it ti it ti it it it it it it Oat S e c t. it n No tt Yes -1 2 o f th e a c id ic e t h e r -s o lu b le m a te r ia l from cocao and bananas (1 1 1 ). a d d itio n , b i o l o g i c a l a c t i v i t y was found a t t h is R f. In By s u it a b le s e le c t i o n o f s o lv e n t s , th e component g iv in g c o lo r t e s t s cou ld be sep a ra ted from th e growth prom oting compound which, n o n e th e le s s , appeared to be IAA. The b e s t so u rces o f in d o le compounds (T able I ) would appear to be s e e d s , f r u i t s , tumor t i s s u e , and some c r u c if e r s (cabbage, c a u liflo w e r , b r o c c o li, e t c . ) . The p h y s io lo g ic a l s ig n if ic a n c e o f IAA in t h e s e t is s u e s i s n o t a t a l l c le a r . I t i s in t e r e s t in g th a t v a r io u s l i n e s o f corn d i f f e r s i g n i f i c a n t l y in regard to th e IAA con ten t in th e seed (143)» and i t i s known th a t h ig h IAA and tryptophan l e v e l s are lin k e d t o th e sugary gene (151)« I t had been su g g ested th a t h igh IAA l e v e l s in immature corn seed were a s s o c ia t e d w ith low n i c o t in ic a c id l e v e l s , due to co m p etitio n fo r tryp top h an , which was th ough t to be th e p recu rsor o f b oth . However, r e c e n t work makes i t appear t h i s i s n ot th e c a s e , s in c e la b e lin g experim ents in d ic a t e th a t tryptophan i s n ot th e p recu rsor o f n i c o t i n ic a c id in corn ( 65)0 I t should be m entioned t h a t th e IAA and tryptophan l e v e l s r i s e to a maximum in th e immature corn seed a t about 18 to 24 days a f t e r p o llin a t io n , and th en th e l e v e l s f a l l w ith approaching m atu rity (143,1 5 1 )* The p h y s io l­ o g ic a l s ig n if ic a n c e o f h ig h l e v e l s o f in d o le compounds in c r u c if e r s i s unknown. L in ser ( 9 6 ) has q u estio n ed any p o s s ib le p h y s io lo g ic a l r o l e o f h igh l e v e l s o f IAN and o th er in d o le compounds in cabbage. In r a p id ly growing s e e d lin g s , where th e growth may be dem onstrated to be under hormonal c o n tr o l by v a rio u s p h y s io lo g ic a l exp erim en ts, i t i s ex trem ely d i f f i c u l t to d e t e c t IAA. I t may be th e m inute amount p r e se n t makes d e t e c tio n im p o ssib le ex cep t by s e n s i t i v e (b u t n o n s p e c if ic ) b io a ss a y s; b u t, on th e o th er hand, as B e n tle y (14) p o in ts o u t, sweeping sta tem en ts a r e n ot y e t pru d en t. -1 3 3* The E x is te n c e o f I n d o le - 3 -A c e tic A cid in Avena C o le o p t ile s I t i s o f i n t e r e s t to retu rn to th e Avena c o l e o p t i l e and summarize th e ev id e n c e a v a ila b le on th e n a tiv e hormone in t h i s organ . I t i s obvious th a t where au xin i s i s o la t e d by a llo w in g i t t o d i f f u s e from th e e x c is e d t i p s in to agar b lo c k s , th a t one i s m easuring p rod u ction p er u n it tim e. I t a ls o appears t h a t some s o r t o f stea d y s t a t e must e x i s t between pro­ d u c tio n and in a c t iv a t io n . The l a t t e r may in v o lv e n o n -s p e c if ic b in d in g , enzym atic d e s tr u c t io n , or some s o r t o f in a c t iv a t io n a t th e a c t iv e s i t e during th e growth p r o c e s s . Though i t i s s ta t e d t h a t c o ld eth er e x tr a c tio n i s a measure o f f r e e au xin in th e t i s s u e , th e evid en ce i s not co n v in cin g . Wildman and Muir (185) cou ld dem onstrate p ro d u ctio n o f IAA (Avena curva­ tu r e t e s t ) from tryptophan during wet eth er e x tr a c tio n o f f e r t i l i z e d tob acco o v a ry s. The p r o c e ss has a low tem perature optimum o f about 13°C, and though th e y s t a t e th e r e was no p rod u ction o f IAA a t 0°C in 16 hou rs, t h e ir d ata in d ic a t e a s l i g h t p rod u ction o f IAA even a t t h is low tem p erature. T erp stra (153)> a ls o u sin g th e Avena cu rvatu re t e s t , found not a l l th e a c id a u x in co u ld be e x tr a c te d in one day a t 4°C from Avena c o l e o p t i l e t i p s , and a f t e r 7 days o f e x t r a c tio n , a c o n sid e r a b le q u a n tity o f n e u tr a l auxin was found. Much more auxin was found a f t e r in cu b a tin g Avena t i p s 1 to 6 days in w a ter. A fte r f r e e z in g and thawing th e c o l e o p t ile t i p s 4 tim e s , l i t t l e a u xin cou ld be d e te c te d , and a ls o g rin d in g fr e sh t i p s reduced th e auxin y i e l d t o a s im ila r e x te n t. I f fr o z e n or fresh -grou n d c o l e o p t il e s were in cu b a ted a t 23°C, more au xin was found, but a d d itio n o f 0 .0 1 ppm o f d ie th y ld ith io c a r b a m a te in h ib ite d t h i s in c r e a s e . I t was su g g e sted th a t d ie th y ld ith io c a r b a m a te i n h i b it s both form ation and d e s tr u c tio n s in c e in exp erim en ts w ith w hole t i p s e x tr a c te d w ith w ater a t 23 °C , i t in h ib it e d d e s tr u c t io n o f a u x in . Thus one can con clu d e t h a t o n ly v ery sh o rt term , -1Alow tem perature e x t r a c tio n s w ith eth e r a re v a lid as e stim a te s o f th e 11f r e e 11 IAA c o n te n t. At l e a s t th r e e e s tim a te s have been made o f th e au xin c o n ten t o f th e Avena t i p (1 -5 mm). Larsen (91) estim a ted 1 .7 x 10“^ jug "IAA e q u iv ." , Thimann (1 5 7 ) su g g e ste d about 1 - 1 .5 x 1 0 " ^ jug "IAA e q u iv ." , w h ile Wildman and Bonner (183) found about 1 .6 x 10- ^ ;ug "IAA e q u iv ." . These e s tim a te s , which a re in rem arkable agreem ent, a re e it h e r based on co ld e th e r e x tr a c tio n or sh o r t term d if f u s io n exp erim en ts. More r e c e n t ly , i t has been e stim a te d th a t th e t i p s u p p lie s an e q u iv a le n t o f about 0 .0 7 5 ml o f IAA ( 6 ,7 ) . M g/ S in c e a 1 mg t i p (2 mm) i s about 0 ,0 0 1 m l, t h i s would correspond to about 7*5 x 10” ^ jug per each t i p , which i s in e x c e lle n t agreem ent w ith th e o th er e s tim a te s . Thus about 10,000 t i p s m ight be a n t ic ip a t e d t o c o n ta in about 0 .7 5 to 1 .0 jxg o f IAA. Wildman and Bonner (183) c o ll e c t e d 23,000 ly o p h y liz e d Avena t i p s (about 2 .3 gm dry w e ig h t), and e x tr a c te d th e m a te r ia l w ith wet e th er in th e c o ld . The eth er e x tr a c ts co n tain ed 2 .3 jag o f "IAA eq u iv ." a s measured by th e Avena cu rv a tu re t e s t . A Salkow ski a s sa y was p o s i t i v e , and in d ic a te d about 1 .8 jag o f "IAA" was p r e s e n t. They a ls o allow ed about 2 0 ,0 0 0 t i p s t o d i f f u s e fo r 3 hours in a sm all volume o f w ater and e s t i ­ mated 1 .9 /ig o f "IAA" by th e Avena cu rvature t e s t , but in t h i s c a se th e S alk ow sk i t e s t was pink in s te a d o f th e u su a l deep red c o lo r . The c o l e o p t il e t i p was found to be 6 tim es as e f f e c t i v e as th e rem ainder o f th e c o l e o p t il e in c o n v er tin g tryptophan to IAA, and th e r e appeared to be r e g e n e r a tio n o f th e c o n v er tin g system a f t e r d e c a p ita tio n o f th e t i p . The m olecu lar w eigh t o f th e d i f f u s i b l e au xin was found to be about 306, but a f t e r e x t r a c tio n in to e th e r , th e m olecu lar w eig h t was estim a ted to be 206. T h is d iscrep a n cy was assumed to be due to th e e lim in a tio n o f d i f f u s i b l e in h ib it o r s by -1 5 e th e r e x t r a c tio n . T erp stra (153) paper chromatographed th e w a ter, t o t a l e th e r , and a c id ic eth er f r a c t io n s from Avena c o le o p t ile t i p s . The a c t i v i t y as determ ined by th e Avena cu rvatu re t e s t corresponded in each ca se to th e Rf o f IAA. Shibaoka and Yamaki (132) allow ed 600 Avena t i p s t o d i f f u s e fo r 3 hours a t 25°C on agar. and th en w a ter. The agar was e x tr a c te d w ith eth er Both th e w ater and eth e r f r a c t io n s were made up in to agar b lo ck s c o n ta in in g 5 x 10”% FeSO^. The l a t t e r s e n s it iz e d th e Avena cu rv a tu re t e s t , r e s u lt in g in a lin e a r range from 0 to 0 .1 jug. Only th e e th e r f r a c t io n was a c t i v e , so th e remainder o f i t was chroma­ tographed in 3 s o lv e n t s , and in each ca se th e Rf corresponded to an Rf o f IAA. Thus from th e a v a ila b le e v id en ce, th e c a se would appear stro n g th a t th e auxin in Avena c o l e o p t il e t i p s i s IAA. 4. Lack o f R e d is tr ib u tio n o f Exogenous I n d o le -3 -A c e tic A cid F ollow in g Geo- or P h o to tro p ic S tim u la tio n Gordon and Eib (51) rep o rted th a t Avena c o l e o p t il e s tr e a te d w ith IAA-2 -C -^ tr a n sp o r te d i t in a p o la r manner a t a v e l o c i t y o f lmm/min. When th e c o l e o p t i l e was illu m in a te d u n i l a t e r a l l y , no d iff e r e n c e in r a d io ­ a c t i v i t y cou ld be d e te c te d between th e l i g h t and dark s id e , but more auxin d id d i f f u s e from th e dark s id e as measured by th e Avena curvature te st. At th e same tim e , Bflnning e t a l . (28) rep o rted s im ila r r e s u l t s . More r e c e n t ly , Ching and Fang (31)» as w e ll as R eisen er and Simon (1 2 7 ), have re p o r ted la c k o f r e d is t r ib u t io n o f r a d io a c tiv e IAA in g e o t r o p ic a lly s tim u la te d r o o ts and c o l e o p t i l e s . I f one a c c e p ts IAA as c o n t r o llin g g e o tr o p ic and p h o to tr o p ic r esp o n se , th en one must assume th a t: ( l ) There i s an e f f e c t on p ro d u ctio n or r e le a s e o f endogenous IAA as a -1 6 r e s u l t o f u n i l a t e r a l g e o tr o p ic or p h o to tr o p ic s tim u la tio n ; (2 ) The in t e r n a l p o o l o f IAA i s sep a ra te from th e e x t e r n a lly su p p lie d p o o l; or (3 ) The su p p lie d IAA sa tu r a te d th e tr a n sp o r t mechanism, le a d in g to f a i l u r e t o r e d is t r ib u t e a d e t e c ta b le q u a n it it y o f IA A -C ^. An exam ination o f t h e s e r e s u lt s in d ic a te s th a t in a t l e a s t some c a s e s th e t h ir d su g g e stio n may n ot a p p ly , so perhaps th e second su g g e stio n i s most a t t r a c t i v e . i s r e d is t r ib u t e d . T h is m ight im ply some complexed or bound form o f IAA Though s e v e r a l bound or complexed forms o f IAA are known, th e p h y s io lo g ic a l s ig n if ic a n c e o f them i s unknown. 5. Bound or Conjugated Forms o f IAA I t was f i r s t observed by Kdgl, E rxleben , and Haagen-Smit (79) th a t a lk a lin e h y d r o ly s is r e le a s e d auxin from s e e d s . S ev e r a l p r o te in s a ls o r e le a s e micro amounts o f auxin fo llo w in g enzym atic a lk a lin e h y d r o ly sis (5 0 ,1 3 0 ) as measured by th e Avena curvatu re t e s t . Though S ie g a l and G a lsto n (133) have dem onstrated a com plexing o f IAA to pea ro o t homogenate p r o t e in , no c r i t i c a l stu d y o f t h i s com plexing has been conducted. Many t i s s u e s w i l l l i b e r a t e au xin in to e th er fo r an extended p erio d o f tim e ( l 6 l ) , and though t h i s was form erly regarded as bound a u x in , i t i s now apparent t h i s li b e r a t i o n r e p r e s e n ts p rod u ction from tryptophan (1 8 5 ). The b e s t known au xin complex i s th a t o ccu rrin g in corn seed and th e n a tu re o f t h i s p recu rso r was e x t e n s iv e ly stu d ied by Berger and Avery ( 1 8 ). These w orkers found th a t aqueous a ceto n e e x tr a c ts o f Country Gentleman sw eet corn seed co n ta in ed a y e llo w , gummy, a q u e o u s-in so lu b le m a te r ia l from w hich, a f t e r a lk a lin e h y d r o ly s is (pH 9 .6 fo r 5 m in .) c r y s t a lin e IAA contam inated w ith a carb on yl c o n ta in in g compound cou ld be i s o la t e d . The m a te r ia l was r e l a t i v e l y s o lu b le in aqueous e th a n o l, m ethanol, dioxan e or d i l u t e a l k a l i , but i t was r e l a t i v e l y in so lu b le in e th e r , hot a c e to n e , or -1 7 d i l u t e a c id . I t was d ia ly z a b le (40$ in 12 hou rs) in crude p r e p a r a tio n s , b u t a f t e r s e v e r a l r e p r e c ip it a t io n s , o n ly 5 -7$ was l o s t upon d i a l y s i s . The p recu rso r co n ta in ed o n ly about 4 .5 $ n itr o g e n , so e it h e r i t was im­ pure or n ot a p r o t e in . S t e h s e l (143) has examined th e n ature o f IAA r e le a s in g m a te r ia l from immature sweet corn seed . T his "auxin complex" was e x tr a c te d by aqueous m ethan ol, d ia ly z a b le , and had an Rf o f 0 .2 5 in butanol/HCl/KLjO ( 6 0 i 25 m l, IN HCl). The m olecu lar w eig h t as determ ined by agar d i f f u s i o n was e stim a ted t o be 300 to 500. Conversion took p la c e r e a d ily upon h e a tin g w ith 0 .0 2 t o 0 .0 0 3 N NaOH fo r f i v e m inutes even under n itr o g e n . The complex was found to g iv e a p o s i t i v e Salkow ski c o lo r , but d if f e r e d somewhat from th a t g iv en by IAA. In co n firm a tio n o f Avery and B erg er, th e "auxin complex" was in a c t iv e in th e Avena cu rvatu re t e s t . Sdding and Raadts (141) rep orted a w a te r -s o lu b le su b stan ce in oat c o l e o p t i l e s w hich appears t o y ie l d IAA (d e te c te d by b io a ss a y ) upon paper chrom atography. T erpstra (153) found a w a te r -s o lu b le complex in Avena c o l e o p t i l e s which y ie ld e d IAA a f t e r a lk a lin e h y d r o ly s is . H ousley and B e n tle y ( 6 9 ) rep o rted t h a t th e w a t e r -s o lu b le , e t h e r - in s o lu b le f r a c t io n in cabbage g iv e s r i s e to an a u xin havin g th e same Rf as IAA in iso p r o ­ panol/am m onia/w ater s o lv e n t , but a low er Rf in n-butanol/am m onia/w ater. I t shou ld be m entioned th a t e a r ly p h y s io lo g ic a l experim ents by Skoog ( 1 3 6 ) u sin g th e deseeded Avena t e s t , and la t e r experim ents by Raadts ( 1 2 3 ) , in d ic a te d a p r esu rso r o f auxin was tr a n s lo c a te d from th e seed to th e c o l e o p t i l e t i p . T his p recu rsor had been su g g e sted by Wildman and Bonner (183) to be tryp top h an , but in d o le a c e ta ld e h y d e has a ls o been su g g e ste d as th e tr a n sp o r te d p recu rsor (1 2 3 ). Pohl (121) and T e g e th o ff ( 1 5 2 ) have s tu d ie d a growth in h ib it o r in corn scu tellu m which i s con­ v e r te d to a u x in by crude homogenates o f Avena c o l e o p t il e t i p s (1 5 2 ). -1 8 The in h ib it o r appears to i n a c t iv a t e IAA by combining w ith i t (1 5 2 ). I t would be a t t r a c t i v e t o suppose t h a t IAA in th e endosperm cou ld be co n v erted to a tra n sp o r ted complex which may be a g a in co n verted to IAA (o r another complex) a t th e c o l e o p t il e t i p . In a d d itio n t o p r o te in s and th e above unknown su b sta n ces which r e ­ l e a s e IAA upon m ild a lk a lin e h y d r o ly s is , s e v e r a l amino a c id co n ju g a tes o f IAA a re known. I t has been known th a t IAA i s excrb ted in u r in e , p a r t ly as th e g ly c in e complex ( 6 0 ) , and r e c e n t ly th e glu tam ine complex has been d e te c te d ( 7 2 ). I n v e s t ig a t io n s by Andreae, Good and a s s o c ia t e s (3 * 4 ,5 * 4 5 ) have shown th a t fe e d in g IAA to s e e d lin g s r e s u lt s in form ation o f th e a s p a r tic a c id co n ju g a te p r im a r ily , though some o f th e IAA was con­ v e r te d to th e am ide. Jepson (71) found th e l a t t e r cou ld be formed, due t o ammonia in th e chromatography s o lv e n t . Andreae e t a l . ex p ressed th e view th a t th e s e r e a c tio n s may be in v o lv ed in d e t o x if ic a t io n . A compound c a lle d a sco r b ig e n has been d e te c te d in v a rio u s c r u c if e r s by Prochazka (1 2 2 ). T his compound y i e l d s IAA upon a lk a lin e h y d r o ly s is , or a sc o r b ic a c id a f t e r a c id h y d r o ly s is . I t i s o f i n t e r e s t t o know i f t h i s compound i s one o f th e unknown com plexes d isc u s se d above. In th e su g g e ste d s tr u c t u r e , th e 3 - in d o le -1 ,2 -p r o p y le n e group i s lin k e d 1 ,2 through o x id e bonds to a s c r o b ic a c id in th e 2 ,3 p o s it io n . 6a Other In d o le Compounds O ccurring in P la n ts Jones and co-w orkers (74) have is o la t e d 3 - in d o le a c e t o n it r ile (IAN) from cabbage and i t has s in c e been d e te c te d in a number o f o th er s p e c ie s (1 9 * 3 0 ,1 0 1 ). I n d o le a c e t o n it r ile i s v ery a c t iv e in th e Avena t e s t , and Thimann (158) has dem onstrated i t s co n v ersio n to in d o le - 3 - a c e t ic a c id . Larsen ( 8 9 ,9 0 ,9 3 ) has d e te c te d 3=in d o le a c e ta ld e h y d e in pea s e e d lin g s -1 9 and Yarnaki and Nakomura (189) rep ort i t to be p r e se n t in corn s e e d lin g s . Gordon (4 9 ) has a ls o d e te c te d i t in d ir e c t ly in p in ea p p le l e a f homogenates and mung bean s e e d lin g s . The iso la tio n o f e th y l IAA from immature corn seed by Redemann, W ittw er and S e l l (124) has been su g g ested to be an a r t i f a c t (74) owing t o th e u se o f eth a n o l during i s o l a t i o n . L ik ew ise, Luckwell and P ow ell (100) have q u estio n ed Teubner's (15^) d e te c tio n o f th e e t h y l e s t e r in ap p le endosperm. The d e t e c tio n o f in d o lep y ru v ic a c id was rep orted by Stowe and Thimann (1 4 5 ). However, i t has been shown th a t s y n th e tic in d o lep y ru v ic a c id breaks down co m p le tely under th e a lk a lin e c o n d itio n s o f chromatography used ( 1 5 97 6 ) . A c h a r a c t e r is t ic p a tte r n o f about s i x breakdown prod u cts a re o b ta in ed upon chromatography in isopropanol/am m onia/w ater. These break­ down p rod u cts have been s tu d ie d by Kaper and V eld stra ( 7 6 ) who fin d 3 n e u tr a l compounds havin g an Rf g r e a te r than IAA; and 3 a c id ic compounds, one o f w hich appears t o be 3 - in d o le g ly c o lic a c id . Though F isc h e r (39) has rep o rted d e te c tio n o f in d o le g ly c o lic a c id in cabbage, B e n tle y (1 4 ) found upon chromatography o f a s y n th e tic sam ple, two s p o ts w ere o b ta in e d . The minor sp o t d id have an Rf about l i k e t h a t rep o rted by F is c h e r , w h ile th e major sp o t had a much lower R f. F isc h e r and co-w orkers ( 4 0 ,1 7 1 9172) as w e ll as Jones and Taylor (73) have rep o rted th e occurren ce o f 3 -in d o le a ld e h y d e in cabbage. The e x is te n c e o f 3 -in d o le c a r b o x y lic a c id in cabbage has been rep o rted by th e l a t t e r a u th o r s. C artw righ t, Sykes and Wain (30) have a ls o found t h i s a c id , along w ith IAN, in chromatograms o f e x t r a c t s o f germ in atin g p e a s. C lark, Dye and Wain (32) found IAA and in d o le c a r b o x y lic a c id in crown g a l l tumors o f tom ato, and a tr a c e o f th e l a t t e r in normal stem s. Faw cett e t a l . (38) have shown th a t upon fe e d in g -2 0 IAN to pea s e e d lin g s , 3 -in d o le a ld e h y d e as w e ll as 3 -in d o le c a r b o x y lic a c id a re found as breakdown products* I t i s su g g ested th a t o^ -oxidation occu rs w ith th e n i t r i l e s in c o n tr a st t o ^ - o x id a tio n o f a c id s and am ides. I t is o f i n t e r e s t th a t IAN i s n o t con verted to IAA in pea s e e d lin g s (150) as measured by e it h e r growth resp on se or enzym atic c o n v ersio n . in t h i s t i s s u e and la c k o f c o n v ersio n I t s d e t e c tio n to IAA, su g g e st s tr o n g ly th a t IAN can n ot be c o n sid e re d a p recu rso r o f IAA. The occu rren ce o f la r g e amounts o f IAN and i t s breakdown p ro d u cts, 3 -in d o lea ld eh y d e and 3 -in d o le c a r b o x y lic a c id , in cabbage su g g e st a s im ila r s it u a t io n in t h is t i s s u e . V arious oth er in d o le compounds are known to occur in a t l e a s t c e r t a in p la n t s p e c ie s . T h eir p h y s io lo g ic a l s ig n if ic a n c e i s unknown, but some o f them co u ld be d egrad ation p rod u cts o f tryptophan and IAA. Compounds rep o rted t o occur a re in d o le (1 4 6 ), N -m ethyl-tryptophan ( 4 4 ,1 4 6 ) , 5-hydroxy- N ,N -d im eth yltryp top h an (1 4 7 ), 5-hydroxytryptam ine (1 1 9 ), 5 -h y d ro x y in d o lea c e t i c a c id (1 6 3 ), and s k a t o le (1 0 4 ). I t i s o f in t e r e s t th a t h y d ro x y la tio n o f th e rin g a ls o o ccu rs upon fe e d in g p h en oxyacetic a c id to p la n ts (1 8 2 ). 7. B io s y n th e s is o f I n d o le -3 -A c e tic Acid Thimann (157) su g g e sted q u ite e a r ly th a t IAA was sy n th e siz e d from tryptophan in Rhizopus su in u s . He had observed th a t tryptophan c o n ta in in g peptone and stro n g a e r a tio n was req u ired fo r good y ie ld s o f IAA from t h is fu n gu s. Other in d ir e c t ev id en ce th a t tryptophan might be con verted to IAA in Avena c o l e o p t i l e s comes from th e e a r ly work o f Skoog ( 1 3 6 ) who observed t h a t tryptophan and tryp tam ine gave a d elayed cu rvatu re in th e d eseed ed Avena cu rvatu re t e s t . The s tu d ie s o f S t e h s e l (143) and T eas, Cameran and Newton (151) on tryptophan l e v e l s and IAA l e v e l s in d ev elo p in g corn seed tended to in d ic a te a c o r r e la t io n between f r e e tryptophan co n ten t and IAA l e v e l s . Both reached a maximum 24 to 28 days a f t e r p o lli n a t io n , -2 1 and f e l l w ith approaching m a tu rity o f th e seed . N its c h (114) has l i k e ­ w is e found a c o r r e la t io n betw een f r e e tryptophan and IAA in th e d evelop ­ in g straw b erry. Lund (102) has found an in v e r se c o r r e la t io n between f r e e tryptophan and IAA in p o llin a t e d tob acco s t y l e s . He s u g g e sts th e p o lle n tu b e s co n v ert f r e e tryptophan o f th e s t y l e to IAA, Other evid en ce f o r a c o r r e la t io n in v iv o has been p resen ted by Yamaki and Nakamura (1 8 9 ). These au th ors re p o rt a c o r r e la t io n e x i s t s between f r e e tryptophan and IAA during g erm in ation o f corn embryos. In endosperms, a very l i t t l e IAA cou ld be d e te c te d , which i s in c o n tr a st to r e s u lt s o f C artw righ t, Sykes and Wain (3 0 ); H ou sley, Booth and P h i l l i p s (? 0 ); and th e p r e se n t in v e s t ig a t o r . A d d itio n a l ev id en ce th a t tryptophan i s con verted to IAA comes from th e work o f Skoog (136) and T su i (162) who found t h a t zin c d e f ic ie n c y r e s u lt s in a low l e v e l o f auxin and tryp top h an . T su i showed th e tryp top h an l e v e l appeared to be a f f e c t e d f i r s t , and th a t th e tr y p to phan-IAA c o n v e r tin g system was j u s t as a c t iv e in th e d e f ic ie n t tomato p la n t s . Skoog has found au xin p rod u ction t o be s e n s i t i v e to X -ir r a d ia - t io n (1 3 5 ) and Weber and Gordon (175) have found th e tryptophan co n v ert­ in g system in v iv o i s q u ite s e n s i t iv e . The in v iv o ev id en ce fo r a c o r r e la t io n betw een tryptophan and IAA i s f a i r l y good. On th e o th er hand, th e l e v e l s o f f r e e tryptophan are fr e q u e n tly two or th r e e ord ers o f m agnitude h ig h er than IAA l e v e l s . Thus i t i s s u r p r is in g th a t IAA l e v e l s appear dependent on f r e e tryptophan l e v e l s . The in v iv o tryptophan fe e d in g experim ents fu r n is h a d d itio n e v i­ dence t h a t tryptophan may be con verted to IAA. The f a c t th a t tryptophan (a s w e ll a s tryp tam in e) g iv e s a growth resp on se in th e deseeded Avena cu rv a tu re t e s t (136) and i s con verted to IAA by Rhizopus (157) has been -2 2 m entioned. I t has a ls o been shown (186) th a t U. zeae myceleum as w e ll as Agrobacterium tu m efacien s (178) convert tryptophan to IAA, Wildman, F e r r i and Bonner (184) showed th a t t r y p to p h a n - in f iltr a t e d spinach l e a f d is k s had a h igh er IAA co n ten t th a t u n in f ilt r a t e d l e a f f i s k s . In d o le ­ p yru vic a c id a ls o was con verted to IAA, but tryptam ine was n o t. Lund (101) has rep o rted th a t fee d in g L-tryptophan to tobacco p o lle n r e s u lt s in an in c r e a s e in th e amount o f IAA in th e p o lle n as d e te c te d by paper chromatography. Dannenburg and Liverman (3 3 ) dem onstrated co n v ersio n o f tryp top han2-C 14 to la b e le d IAA in immature w aterm ellon s l i c e s . F iv e r a d io a c tiv e E h rlich p o s i t i v e a c id ic compounds were d e te c te d , one o f which was IAA, I t was su g g ested one o f th e o th ers was some r e s id u a l in d o lep y ru v ic a c id w hich f a i l e d to breakdown fo llo w in g a lk a lin e chromatography. Two b a s ic compounds were found, one o f which was su g g ested to be tryptam ine; and two n e u tr a l compounds were d e te c te d , one o f which appeared t o be IAN. In a d d itio n to th e above in v iv o fe e d in g exp erim en ts, Wildman and Muir (185) have dem onstrated co n v ersio n o f tryptophan to auxin during e th e r e x tr a c tio n o f tob acco o v a ry s. The e x is t e n c e o f 3 -in d o le a c e ta ld e h y d e , a supposed in ter m ed ia te in th e tryptophan-IAA c o n v er sio n , in a t l e a s t some p la n t s , has a lrea d y been d is c u s s e d . Though in d o le p y r u v ic a c id has not y e t been d e te c te d d i r e c t l y , ammonical chromatography breakdown p roducts observed a f t e r fe e d in g tr y p to ­ phan to watermelon s l i c e s ( 7 6 ) , c l o s e l y resem ble breakdown prod u cts o f 3 - in d o lep y ru v ic a c id . Tryptamine a ls o occurs n a tu r a lly in some p la n ts (1 8 0 ). There have been many ,in v it r o d em onstrations o f co n v ersio n o f tr y p to ­ phan to IAA by v a r io u s p la n t t i s s u e s (4 9 ,6 3 ,1 0 2 ,1 8 3 ,1 8 4 ,1 8 9 ) . Wildman, F e r r i and Bonner (184) found co n v ersio n in spinach l e a f homogenates o f -2 3 L -tryptophan t o IAA, The pH optimum was 7*5, oxygen was req u ir e d , and co n v e r sio n was s e n s i t i v e t o cyan id e or b i s u l f i t e . t h a t an aldehyde m ight be an in te r m e d ia te , The l a t t e r su g g ested Gordon and Sanchez (4 7 ,4 8 ) found homogenates o f p in ea p p le l e a f b a ses could co n v ert tryptoph an , try p ta m in e, 3 - in d o le p y r u v ic a c id , or 3 -in d o le a c e ta ld e h y d e to IAA. Gordon (4 9 ) a ls o rep o rted th a t mung bean homogenates con vert in d o lep y ru v ic a c id , but n o t tryp tam in e, to IAA. Wolf (186) f a i l e d to f in d co n v ersio n o f tryp tam in e by U s tila g o zeae c u ltu r e s . Yamaki and Nakumura (189) r e ­ p o r te d co n v ersio n o f tryp tam ine by corn embryo p r e p a r a tio n s, though i t co u ld not be deom onstrated in th e p resen t work th a t corn s e e d lin g s cou ld co n v ert tryptam ine to IAA. These l a t t e r workers a ls o found c o n v ersio n o f in d o le a c e ta ld e h y d e to IAA by corn embryo hom ogenates. Henderson ( 6 3 ) has d e te c te d tryptophan-IAA co n v ersio n in sun flow er stem and crown g a l l homo­ g e n a te s. The l a t t e r had a h ig h er co n v ertin g a c t i v i t y . When normal stem p re p a r a tio n s were mixed w ith crown g a l l p r e p a r a tio n s , th e a c t i v i t y o f th e l a t t e r was d ep ressed . S in c e th e r e appeared to be no d if f e r e n c e in de­ s t r u c t io n , th e p resen ce o f an in h ib it o r in normal stem t i s s u e was su g g e st­ ed . The pH optimum was between 7*0 to 8 .0 . Wildman and Bonner (183) have r ep o rted Avena c o l e o p t i l e t i p s con vert tryptophan to IAA s i x tim es as e f f e c t i v e l y as b a sa l p o r tio n s o f c o l e o p t ile s on a fr e s h w eigh t b a s is . A ls o , th e r e appeared to be r eg en e r a tio n o f th e co n v ertin g a b i l i t y in de­ c a p ita te d c o l e o p t i l e s . Gordon has stu d ie d th e in t e r c e l l u l a r l o c a l i z a t i o n o f th e tryptophan-IAA c o n v er tin g system in mung bean s e e d lin g s (46) and r e p o r ts a l l th e a c t i v i t y r e s id e s in th e s o lu b le f r a c t io n . He found t h is was tr u e when th e g rin d in g medium was is o t o n ic su ero se-p h o sp h a te, w ater o r i s o t o n ic Carbowax 4 ,0 0 0 . He a ls o found th e s o - c a lle d F r a c tio n I p ro- -2 4 t e i n was i n a c t i v e , whereas th e c o n v ertin g a c t i v i t y r e s id e d in th e h e te r ­ ogeneous cy to p la sm ic p r o t e in s . In t h is stu d y , IAA was is o la t e d by paper chromatography o f an a c id e th e r e x tr a c t o f th e in cu b a tio n m ixtu re. i s o l a t e d IAA was a ssa y ed by th e S alkow ski and Avena cu rv a tu re. The These r e ­ s u l t s a re in c o n tr a s t to th o s e o f th e p resen t stu d y w ith corn s e e d lin g s . Bonner (21) comments th a t a t l e a s t some tryptophan-IAA co n v ertin g a c t i v i t y i s found in most t i s s u e s , even though on p h y s io lo g ic a l grounds one would ex p ect none. D ir e c t in v iv o la b e lin g experim ents showing try p ­ tophan as th e o n ly sou rce o f IAA have n ot y e t been p o s s ib le . However, i t can be concluded th a t tryptophan may be con verted in v iv o to IAA. In summary, th e b io s y n t h e s is o f IAA from tryptophan in s o fa r as i t i s known in p la n ts (oxygen requirem ent and CN s e n s i t i v i t y ) , would c o r r e s ­ pond t o an amino a c id o x id a se . Krebs (86) observed D- and L-amino a c id o x id a se a c t i v i t y in l i v e r and kid ney p rep a ra tio n s from a number o f a n im a ls. He dem onstrated se p a r a tio n o f D- and L-amino a c id o x id a se a c t i ­ v i t y , and found th e L-amino a c id o x id a se was s e n s i t iv e to cy a n id e. D-amino a c id o x id a se was found to be an FAD f la v o p r o t e in . The More r e c e n t ly ( 1 3 4 ), th e L-amino a c id o x id a se has a ls o been shown to be a fla v o p r o te in co n ta in in g FMN. Tryptophan is* one o f th e amino a c id s most r e a d ily o x i­ d iz ed by enzymes from v a r io u s anim al or m ic r o b ia l s o u r c e s. formed a s an in te r m e d ia te i s decomposed by th e o f th e reduced f l a v i n w ith oxygen. ^2P z The k eto a c id formed from r e a c tio n However, amino a c id o x id a se a c t i v i t y in h ig h e r p la n ts has n ot y e t been d e te c te d (1 4 8 ), u n le s s th e tryp top h an IAA system i s in f a c t such a system . I t would be o f i n t e r e s t to know i f th e tryptophan-IA A system cou ld a ls o con vert oth er amino a c id s . -2 5 Bo S y n th e tic P la n t Growth S ubstances Many d iv e r s e s y n th e tic compounds have been found t o have a c t i v i t y as growth su b sta n ces in v a r io u s b io lo g i c a l t e s t s . Kflgl and Kostermans ( 8 3 ) rep o rted in 1935 th a t th e m ethyl IAA and in d o lep y ru v ic a c id were a c t i v e , but i n d o l e l a c t i c a c id was in a c t iv e , in th e Avena cu rvatu re t e s t . Haagen-Smit and Went ( 5 8 ) a ls o rep orted th e a c t i v i t y o f in d o lep y ru v ic a t about th e same tim e . Thimann (158) found in d e n e -3 -a c e tic had s lig h t a c t i v i t y but c o m u r a l-2 -a c e tic a c id had no a c t i v i t y in th e Avena s e c tio n te st. The work o f Zimmerman and a s s o c ia t e s both broadened th e scope o f a c t iv e compounds and in trod u ced th e f i r s t p r a c t ic a l u se fo r a s y n th e tic a u x in . In 1935* Zimmerman and W ilcoxon (193) and H itchcock (66) rep o rted p h e n y la c e t ic , Y -p h en y lp ro p io n ic, in d o le p r o p io n ic and naphthylene a c e t ic a c id s were a c t i v e in in d u cin g r o o tin g o f c u t t in g s , as w e ll as ind ucin g oth er fo rm a tiv e or m o rp h o lo gical r e s p o n se s . In th e same y e a r , Thimann and K o e p fli (159) rep o rted IAA was a c t iv e in th e r o o tin g o f c u t t in g s . Thimann and Went ( l 6 l a ) had a lrea d y stu d ie d th e nature o f th e ro o t form ing hormone and concluded i t was id e n t i c a l to au xin . Zimmerman and H itchcock (1 9 0 ,1 9 1 s1 9 2 ) , u sin g th e m orph ological and e p in a s t ic resp o n ses o f th e in t a c t p la n t as a primary c r i t e r i a o f a c t i v i t y , soon added h a lo g e n a te d -p h e n y la c e tic , -b e n z o ic , and -p h en o x y a cetic a c id s t o th e l i s t o f s y n t h e t ic growth r e g u la t o r s . Many compounds showed a c t i v i t y on in t a c t p la n ts even though no a c t i v i t y was shown in c l a s s i c a l growth t e s t s . Even e th y le n e was a c t iv e in producing t y p ic a l e p in a s t ic and "form ative" re sp o n se s o f in t a c t p la n t s . I t was a lr e a d y apparent th a t v a rio u s t e s t s le d to d i f f e r e n t e s tim a te s o f th e a c t i v i t y o f th e same compound. -2 6 - G u stafson (53) found in 1938 th a t s y n th e tic "auxins" such as in d o le p r o p io n ic a c id , in d o le b u ty r ic a c id and p h e n y la c e tic a c id induced p arthen oca r p ic f r u i t in tom ato. Zimmerman and H itchcock (191) observed in crea sed f r u i t s e t in tomato a f t e r treatm ent w ith a number o f s y n th e tic "auxins." At th e p r e se n t tim e , com mercial u se o f s y n th e tic growth su b sta n ces fo r f r u i t s e t and p a rth en o ca rp ic f r u i t i s lim it e d to th e f i g ( 1 4 4 ), where normal p o llin a t io n i s accom plished by a sm all wasp which o v u la te s in male flo w e r s produced by a sep a ra te male t r e e . Another p ro p erty o f auxin was found by Laibach (88) in th a t auxin seemed to p la y a r o l e in a b c is s io n . LaRue (94) dem onstrated d elayed a b c is s io n in c o le u s by s y n th e tic growth su b sta n c e s, and th e lit e r e a t u r e in th e f i e l d has been review ed by A d d ico tt and Lynch ( 2 ) . Commercial u se in p r ev e n tin g p r e -h a r v e st drop, e s p e c ia lly in a p p le s, i s f a i r l y w ide spread . A lthough th e above u se s o f s y n t h e t ic p la n t growth su b sta n ces are c o n s id e r a b le , th e s e l e c t i v e h e r b ic id a l e f f e c t s o f t h e s e compounds are o f enormous economic im portance in a g r ic u ltu r e . The s e l e c t i v e p h y t o t o x ic ity o f s y n th e tic "auxins" and o th er org a n ic compounds was in v e s t ig a t e d in England by S la d e , Templeman and Sexton (139) as w e ll as Nutman, Thornton and Q u a stel (1 1 6 ); and by a resea rch team le d by Kraus, Norman, M inarik and o th e r s (8 5 ) in th e U n ited S t a t e s . The fin d in g th a t im portant grain crops t o le r a t e d q u a n t it ie s o f th e s e m a te r ia ls which k i l l e d many d ico ty led o n o u s s p e c ie s , made them extrem ely v a lu a b le . In a d d itio n , many o th er p h y to to x ic org a n ic ch em ica ls have now been p la ced in commercial u se as a d ir e c t r e s u lt o f th e in t e n s iv e work stim u la te d by th e i n i t i a l d isc o v e r y . In a d d itio n to th e p r a c t ic a l a s p e c t s , th e s y n th e tic growth su b sta n ces have been o f g rea t t h e o r e t ic a l i n t e r e s t to p la n t p h y s i o lo g is t s . In t h is -2 7 reg a rd , g rea t e f f o r t has gone in to attem p ts t o d e fin e th e growth in d u cin g s i t e in terms o f th e s p e c i f i c s tr u c t u r a l requirem ents n e c e s sa r y , or to c o r r e la t e in v iv o a c t i v i t y w ith some model system . These ty p e s o f s tu d ie s have le d t o fin d in g a c t i v i t y in d iv e r s e , unexpected groups o f compounds; but on th e o th er hand, w ith in a r e la t e d group o f compounds, c o m p le te ly in a c t iv e an alogu es a re common. I t i s now apparent th a t many secondary f a c to r s o p era te in th e in vivo t e s t s . A compound may n ot be tak en up, or i t may be in a c tiv a t e d by p h y s ic a l a d so r p tio n , m etab olic c o n ju g a tio n , or d e s tr u c tio n . At l e a s t th e p o t e n t i a l l y a c t iv e compound does not reach th e a c t iv e s i t e . C on versely, an in a c t iv e compound may be con verted t o an a c t iv e d e r iv a t iv e w ith in th e p la n t (1 7 4 ). The u n resolved problem o f q u a l i t a t i v e d if f e r e n c e s in a c t i v i t y in d if f e r e n t b io a ss a y s may in p a rt be due to th e s e secondary f a c t o r s , but cou ld a ls o be due t o more than one a c t i v e s i t e . A more d e t a ile d d is c u s s io n o f c o m p e titiv e in h ib it io n , as w e ll as in h ib it io n o f tro p ism s by s y n th e tic p la n t growth su b sta n c e s, w i l l be g iv en in th e in tr o d u c tio n to th e papers on th e s e t o p ic s . -2 8 - EXPER IMEKTAL I. STUDIES ON PLANT GROWTH SUBSTANCES A. O ccurrence and B io s y n th e s is o f I n d o le -3 -A c e tic A cid 1. In V itro Experim ents on th e B io s y n th e s is o f IAA from Tryptophan In p r elim in a r y ex p erim en ts, A s p e r g illu s n ig e r , Rhizopus n ig r ic a n s and P e n ic iliu m chrysogenum were examined fo r t h e ir a b i l i t y to produce IAA from tryp top h an . I t was hoped t h a t a h ig h ly a c t iv e sou rce o f th e co n v ertin g system cou ld be found. The above fu n g i were grown on liq u id p o ta to d e x tr o se media fo r te n days in one l i t e r f l a s k s . The mats were then cu t in h a lf and tr a n s fe r r e d to 200 ml 0 .0 0 5 M p otassiu m phosphate b u ffe r pH 7 .0 p lu s or minus 0 .5 mg/ml D L-tryptophan. The s o lu t io n s were v ig o r o u s ly a era ted fo r 12 hou rs, and 2 ml sam ples examined by Salkow ski a ssa y (1 4 9 ). A t e s t fo r Salkow ski p o s it iv e su b sta n ces was g iv en by Rhizopus n ig r ic a n s and P e n ic iliu m chrysogenum, w ith th e former showing th e most a c t i v i t y . Some a d d itio n a l experim ents w ith Rhizopus mats in d ic a te d a maximum c o lo r (Salkow ski a ssa y ) o f about 17°5 Mg o f "IAA e q u iv a le n ts" was reached in about 24 hours, A crude homogenate was prepared by g rin d in g 5 grams ( fr e s h w eig h t) o f fro z e n Rhizopus mat in 30 ml o f 0 .0 1 M p otassium phosphate b u ffe r pH 7*0 a t 1°C u sin g a g la s s hom ogenizer. A fte r pouring through s e v e r a l la y e r s o f c h e e se c l o t h , th e f i l t r a t e was c e n tr ifu g e d a t 1°C in an an gle c e n t r i­ fu g e a t 2 ,0 0 0 x G fo r f i v e m in u tes. The sup ernatan t liq u id was used as a sou rce o f th e crude enzyme. An a s sa y m ixture c o n s is te d o f 2 ml crude enzyme, 2 ml o f 0 .0 0 5 M tryptophan (10 MgClg- jjM) , 2 ml o f 0 .0 0 5 M sodium p y ru v a te, and 0 .5 ml o f 0 .0 1 M A 2 ml sample was d e p r o te in iz e d a f t e r 0 , 75 and 210 m inutes w ith -2 9 1 ml o f 5$ TCA. Salkow ski a s sa y on 2 ml o f d e p r o te in iz e d supernatant in d ic a te d th a t a v ery sm all amount o f IAA. co u ld be d e te c te d a f t e r 75 and 210 m in u tes. B o iled enzyme and no-tryptophan c o n tr o ls were n e g a tiv e . P rev io u s in cu b a tio n o f th e mat on tr y p to p h a n -co n ta in in g media gave no ev id en ce f o r a d a p tiv e form ation o f th e "enzyme". The om ission o f MgCl2 or p yru vate s e p a r a te ly had no e f f e c t on a c t i v i t y . A lthough v i s u a l l y d e t e c ta b le pink c o lo r appeared in th e above a ssa y s a f t e r ±i to 3 t hours in cu b a tio n a t 30°C, o n ly a f t e r 18 to 20 hours was th e Salkow ski c o lo r r e a d ily m easurable w ith th e p h o to e le c tr ic c o lo r im e te r . A fte r p rolon ged in c u b a tio n , IAA cou ld be d e te c te d by paper chroma­ tography ( isoprop an ol/am m on ia/w ater, 1 0 ; l s l ascen d in g) o f th e eth er e x t r a c ts o f a c i d i f i e d d e p r o te in iz e d su p ern a ta n ts. However, when TCA was used f o r d e p r o t e in iz a t io n , a y e llo w sp o t having th e Rf o f IAA appeared on paper chromatograms when sprayed w ith E h r lic h 's reagen t (1$ p -d im eth y lam inobenzaldehyde and 8 .5 $ HC1 in 95$ e th a n o l). z a tio n by b o ilin g in d ic a te d th e p resen ce o f IAA. S u b seq u en tly, d e p r o te in i­ However, when in d o le - 3- a c e t o n i t r i l e and tryp tam in e were used as s u b s tr a te s , l i t t l e i f any IAA could be d e te c te d by d ir e c t Salkow ski a ssa y on paper chromatography a s d e sc r ib e d above. In s e v e r a l exp erim ents, sm all samples o f th e super­ n a ta n ts were a ls o chromatographed on paper. There appeared to be a p o s s ib le in te r -c o n v e r s io n o f tryptophan and tryptam ine in s e v e r a l o f th e exp erim en ts. A lso , th e r e was a marked d isap pearan ce o f IAN. Corn s e e d lin g s (W23 x 0h51A) were found to be as a c t iv e a source o f th e tryptophan co n v er tin g crude enzyme as R hizopus, so fu r th e r s tu d ie s were w ith t h i s t i s s u e . For th e s e a s s a y s , 5 gm ( fr e s h w eig h t) o f corn was ground in 30 ml o f 0 .0 1 M p otassium phosphate b u ffe r . a re as p r e v io u s ly d e sc r ib e d fo r R hizopus. The p rep a ra tio n and a ssa y I t was found th a t a c t i v i t y in -3 0 corn was s e n s i t i v e t o f r e e z in g (T able I ) and t h a t r o o ts were a s a c t iv e a s s h o o ts . TABLE I O p tic a l D e n sity a t 525 nm o f 2 ml D ep ro tein ized Supernatant and 8 ml S alkow ski R eagent A fte r 1^ Hours In cu b ation o f Tryptophan w ith Frozen and Unfrozen Corn Homogenate P rep a ra tio n s Homogenate S u b stra te ^ -d a y -o ld sh o o ts 10 juM tryptophan 0 .0 8 6 ^ -d a y -o ld sh o o ts 0 0 .0 0 5 4 - d ay-oId r o o ts 10 O.D. 525 0.0^6 jjM tryptophan ^ -d a y -o ld r o o ts 0 0.002 R oots fro z e n 2X 10 jiH tryptophan 0.000 Roots fro z en 2X 0 0 .0 0 1 Some corn homogenate prepared as p r e v io u s ly d escrib ed was c e n t r i­ fuged 1 hour a t 25*000 x G. The o r ig in a l enzyme, th e su p ern a ta n t, and th e r e s id u e (resuspended in th e o r ig in a l volume) were compared. A ll th e a c t i v i t y was found in th e r e s id u e , so i t appeared th e co n v ertin g flen­ zyme" was p a r t ic u la t e in n a tu re. The above two o b se r v a tio n s ( p a r t ic u la t e n ature and s e n s i t i v i t y to fr e e z in g ) made i t d e s ir a b le t o examine th e p a r t ic u la t e f r a c tio n s under is o t o n ic c o n d itio n s . The procedure o f M illerd et a l. (107) was u sed. T h ir ty grams o f t i s s u e (F.W .) was ground a t 1°C in a mortar w ith 10 gms o f a n a ly t ic a l grade sea sand p lu s ^0 ml o f 0.1M potassiu m phosphate b u ffe r pH 7 .0 , and O.^M su c ro se i s o l a t i o n s o lu t io n . The d eb r is was s tr a in e d through 8 la y e r s o f c h eese c lo t h , th e f i l t r a t e c e n tr ifu g e d a t 5 0 0 x G fo r 5 m in u tes, and th e r e s id u e resuspended in 35 ml o f i s o l a t i o n s o lu t io n . The sup ernatan t liq u id was c e n tr ifu g e d a t 1 0 ,0 0 0 x G fo r 15 m inutes and th e r e s id u e (m itoch on d rial fr a c tio n ) resuspended in 20 ml o f -3 1 i s o l a t i o n s o lu tio n * The resuspended r e sid u e was c e n tr ifu g e d again fo r 15 m inutes a t 1 0 ,0 0 0 x G and th e p r e c ip it a t e d m a te r ia l resuspended in 3 .5 ml o f i s o l a t i o n so lu tio n * The r e s u l t s o f s e v e r a l experim ents a re summarized in Table I I . In a l l c a s e s , th e r e s id u e and th e sup ernatan t were r e l a t i v e l y in a c t iv e , but i t must be p o in te d out 5 -7 ml o f supernatant would be req u ired to be eq uiv­ a le n t to 0*5 ml o f m itochondria or r e s id u e (1 /7 o f th e t o t a l volume) on a f r e s h w eig h t b a s is . A ls o , th e supernatant developed a red-brown c o lo r w ith S alk ow sk i r e a g e n t, making d e t e c tio n o f l e s s than 5-10 ^g IAA tube im p o ssib le by th e d ir e c t Salkow ski a ssa y . Sodium p yru vate, p (-k e to g lu ta r a te and p y r i- d oxal phosphate do not s tim u la te th e a c t i v i t y . TABLE I I A c t i v i t y o f R e sid u a l, M ito ch o n d ria l, and Supernatant F r a c tio n s From 4-Day-Old Corn Shoots^ 1 . Exp. N o.; R esidue (500 x G) Salkow ski C olor Complex P .P . a t 525 mu*' 24 30 . __ 3 1 ..... 32 33 0 .0 3 8 0.033 0 .0 3 3 0.018 CM F r a c tio n 2 . M itochondria (1 0 ,0 0 0 x G) 0 .0 8 4 0 .0 9 4 0.103 0*104 0 .1 6 3 0 .1 5 0 3 . Supernatant 4. M ito. + © <~ketoglutarate in s te a d o f pyruvate 0*040 0 .000 —— ------- - 0 .0 2 0 0 .0 1 0 0 .0 9 8 0 .1 1 4 0 .1 2 5 0 .1 6 4 0 .1 0 1 ------- 0*114 0 .1 1 8 0 .1 4 8 0 .1 2 4 0 .1 4 4 0*144 5- #4 p lu s p y r id o x a l phosphate^ 6. #2 p lu s p y r id o x a l phosphate-^ 7. #2 minus p yruvate 0 .098 — ------ - 0*103 ------ -4?he a s sa y m ixture c o n s is te d o f 2 ml o f 0 .0 0 5 M tryptophan; 2 ml o f 0 ,0 0 5 M sodium p yru vate; 0 .5 ml o f 0 .1 M MgClg; 0*5 ml o f r e s id u e or m itochondria or 2 .0 ml sup ernatan t; and su c r o se (0.4M) phosphate (0 .1 M) b u ffe r to b rin g th e t o t a l volume to 6 .5 m l. The in cu b a tio n p erio d was 8 hours a t 30°C. p 0.D* w ith tryptophan minus O.D. w ith o u t tryptophan* ^ 0 . 0 5 JuM. -3 2 Two o th e r c o - f a c t o r experim ents were run u sin g a sm a ller volume a ssa y m ixtu re (T able I I I ) . TABLE I I I E f f e c t o f V arious F a cto rs on th e Tryptophan C onverting System o f 4-Day-Old Corn Roots and S h o o tsi "Enzyme*1 Estim ated IAA Equiv. S u b stra te jug/tube ( 2 .5 ml) F actors M issing___________ 1________2______ 1. S h oots M itochondria 0 2. Shoots M itochondria B o ile d 0 3. Shoots M itochondria Pyruvat e , S u ec. 1 6 .7 8 .0 4. Shoots M itochondria ATP 1 6 .7 2 9 .0 5. Shoots M itochondria ATP, P y r ., Succ. 2 3 .4 2 9 .0 6. S h oots M itochondria Sucrose 2 5 .0 1 8 .0 7* S h oots R esidue 0 1 6 .3 5 .0 8. Supernatant 0 9. R oots M itochondria 0 2 6 .5 2 8 .0 10. Roots M itochondria B o ile d 0 0 .0 0 .0 11. R oots M itochondria P y r ., S u cc. 2 6 .5 2 9 .0 12. R oots M itochondria ATP 1 1 .6 3 6 .0 13. Roots M itochondria P y r ., S u c c ., ATP 1 6 .7 3 0 .0 14. Roots M itochondria Sucrose 1 8 .3 3 0 .0 15. R oots R esidue 0 4 0 .0 5 .0 16. R oots Supernatant 0 ------- ------- oo o 1 3 .3 1 2 .0 0 .0 ■^Assay c o n s is t e d o f 0 .5 ml "enzyme*1, 0 .2 ml ATP 10" M, 0 .2 5 ml MgCl2 0 .0 1 M, 0 .2 5 ml sodium pyru vate 0 .0 0 5 M, 0 .2 5 ml potassium su c c in a te 0 .0 0 1 M, and 0 .5 ml tryptophan 0 .0 0 5 M. Sucrose was added to bring m o la r ity o f m ixtu re to 0 .5 M and the t o t a l a ssa y volume was 2 .5 ml. -3 3 I t i s apparent th a t ATP, p y ru v a te, s u c c in a te or s o lu t io n s made is o t o n ic w ith su c r o se a re n o t s tim u la to r y . The high a c t i v i t y in th e r e s id u e f r a c t io n (500 x G, 5 m in .) in Experiment 1 probably in d ic a te s a h ig h er c e n t r ifu g a t io n speed , b rin g in g about d e p o s itio n o f some m itochondria in t h i s f r a c t io n . S in c e i t appeared th a t none o f th e c o - f a c t o r s in clu d ed were e s s e n t i a l , o th e r a ssa y s o f th e su p ern a ta n t, r e s id u e and m ito ch o n d ria l f r a c t io n s were run on an e q u iv a le n t fr e s h w e ig h t b a s is (3 gms per t u b e ) , o m ittin g every­ th in g e x cep t MgCl£ and tryp top h an . IAA was added to n o-tryptophan c o n tr o l tu b es e it h e r b e fo r e in c u b a tio n or a f t e r in cu b a tio n and d e p r o te in iz a tio n . These r e s u l t s a r e shown in T able IV. TABLE IV The R e la tiv e Salkow ski A c t iv it y o f th e Whole Homogenate S u p ern atan t, M itoch on d rial and R esid u al F r a ctio n s From Corn S h oots When Incubated w ith Tryptophan, or When IAA was Added^ o 0D Whole Homogenate .095 Supernatant . 064 M ito ch o n d ria l (1 0 ,0 0 0 x G) R esidue (500 x G) .010 .009 .073 .209 . 546 .050 .024 .016 .075 .208 .537 .062 .223 .------- .035 . 2 64 .492 .801 .0 3 1 .188 ------ ------ ------ ------ * O OO F r a c tio n OD2 fo r IAA Added to No-Tryptophan C on trols I n itia lly A fte r In cu b ation 8 Hours 30°C 1 0 0 JLLK 5 jug 10ju£.. 25 Mg 50 Mg 100 Mg ^Assay m ixture = 4 ml enzyme (3 gins - 37 s h o o t s ), £ 1 ml o f L -tryptophan (5 mM), - 1 ml w a te r, + 0 .5 ml o f 0 .0 1 M MgCl2 . Two ml was d e p r o te in iz e d by b o ilin g and a ssa y ed w ith 8 ml o f Salkow ski r e a g en t. o In each c a s e , th e o p t ic a l d e n s it y o f a no-tryptop han c o n tr o l w ith ou t added IAA has been su b tr a c te d . The data p re sen te d in Table IV i l l u s t r a t e s th e im p o s s ib ilit y o f d ir e c t S alkow ski a ssa y f o r sm a ll amounts o f IAA in th e p resen ce o f h igh concen- -3 4 - t r a t io n s o f sup ernatan t or w hole homogenate f r a c t io n s . The in c r e a s e in th e o p t ic a l d e n s it y o f th e whole homogenate and supernatant f r a c t io n s over th e n o-tryp top h an c o n tr o ls su g g e sts c o n sid e r a b le a c t i v i t y might r e s id e in th e s e f r a c t io n s a l s o . D e str u c tio n or in a c t iv a t io n o f IAA a ls o may have been a f a c to r in th e s e t e s t s s in c e when 100 jug IAA was added b e fo r e in c u b a tio n , c o n sid e r a b le apparent d e s tr u c tio n occu rred . T his apparent d e s tr u c tio n was o f th e same order o f m agnitude (about 80$) in a l l f r a c t io n s c o n sid e r in g th e i n s e n s i t i v i t y o f th e Salkow ski a ssa y w ith th e w hole homogenates and supernatant f r a c t io n s . R oots and imbibed k e r n e ls were fr a c tio n a te d as above and th e equiva­ l e n t o f 15*6 gm o f r o o ts or k er n e ls incubated w ith 10 juM o f L-tryptophan in a t o t a l volume o f 8 ml (T able V) . TABLE V C onverting A c t i v i t y o f V arious F r a c tio n s o f Corn R oots and K ernels when Incubated fo r 8 Hours With and W ithout Tryptophan F r a c tio n Roots P .P .________________ ^Tryptophan -Tryptophan D if f , Imbibed K ernels P .P .____ +Tryptophan -Tryptophan D if f . Whole Homogenate .2 7 2 .260 .012 *272 .240 .0 3 2 Supernatant .2 0 1 .1 7 4 .0 2 7 .149 *097 .0 52 R esidue (500 x G) .0 7 6 .0 4 1 *035 M itochondria (1 0 ,0 0 0 x G) .190 .061 .129 — *377 - .248 .1 2 9 A gain in t h i s experim ent i t would appear th a t th e m itochondrial f r a c tio n c o n ta in s most o f th e c o n v er tin g a c t i v i t y . However, o n ly in th e k er n e l super­ n a ta n t was th e no-tryp top h an o p t ic a l d e n s ity f a i r l y low . The d isap pearance o f IAA during in c u b a tio n , and th e a b i l i t y to d e t e c t IAA w ith Salkow ski -3 5 r e a g en t in v a r io u s f r a c t io n s , was not determ ined. S in c e a c t i v i t y in th e w hole homogenate i s low , i t i s p robable th e supernatant i s e it h e r d es­ tr o y in g IAA or p r e c lu d in g d e t e c tio n . In th e above experim ents em ploying th e Salkow ski a ssa y procedure to d e p r o te in iz e d in cu b a tio n m ix tu res, i t was im p o ssib le to d ecid e whether th e m ito ch o n d ria l f r a c t io n accounted fo r most o f th e IAA b io s y n th e s is from tryp top h an . I t i s however c e r t a in th a t i s a c t iv e in th e c o n v e rsio n th e m itoch on d ial f r a c tio n o f tryptophan to IAA. I t was o f i n t e r e s t to know how th e whole homogenate and supernatant in a c t iv a t e s or d e s tr o y s IAA. In two exp erim en ts, th e supernatant f r a c tio n from corn sh o o ts was in cu b ated w ith 40 jug o f IAA a t 30°C, e it h e r w ith or w ith o u t added c a t a l a s e . (The c a t a la s e was o b tain ed from W orthington and 170 u n it s used p er tu b e , 1 u n it = 1 mg HgOg/Min). Samples were d e p r o te in iz e d and examined by Salkow ski a ssa y a t 0 , 2 , 4 and 8 h ou rs. The r e s u lt s in d i­ ca ted about ?5$ o f th e IAA d isap p eared even when th e tu b es were im m ediately d e p r o te in iz e d . The rem aining IAA d id not d isap p ear upon in c u b a tio n , and th e a d d itio n o f c a t a la s e had no e ffe c t. S e v e r a l experim ents were run in w hich, a f t e r a d d itio n o f 5» 1 0 1 20 and 40 jug o f IAA to th e v a rio u s f r a c t io n s , th e a s sa y m ixtu res were im m ediately d e p r o te in iz e d by s a tu r a tio n w ith ammonium s u lfa te . The IAA was i s o l a t e d by paper chromatography o f th e a c id eth er f r a c t io n s and t h e chromatograms dipped in E h r lic h 's r e a g en t. An attem pt was made to e lu t e th e p u rp le sp o ts w ith 2 ml o f 5°$ dioxane p lu s 4 drops o f HC1. Even though t h i s method d id n ot le a d to s t r i c t q u a n tit a tiv e r e s u l t s , i t in d ic a te d r e l a t i v e l y g rea t l o s s e s when IAA was added to whole homogenates o f corn s h o o ts , r o o ts or k e r n e ls . Thus i t was concluded th a t IAA appeared to be adsorbed on th e p r o te in o f th e crude w hole homogenate or su p ern a ten t f r a c t io n s , making i t im p o ssib le t o d i r e c t l y compare th e -3 6 r e l a t i v e a c t i v i t y o f th e s e f r a c t io n s w ith th e m ito ch o n d ria l f r a c t io n , from which r e c o v e r ie s were h ig h e r . The a d so rp tio n o f IAA to p r o te in o f pea sh o o t homogenates has been rep orted to occur by Marre (163) w ith ou t th e p r esen ce o f ATP a s was rep o rted p r e v io u s ly by S ie g e l and G alston (133) i*or crude pea r o o t hom ogenates. In a d d itio n , Brian ( 2 5 ) has d i s ­ cu ssed th e a d so r p tio n o f 2 -m eth y l-4 -ch lo ro p h en o x y a cetic a c id to f i l t e r e d crude homogenates o f v a r io u s p la n t s p e c ie s , u sin g monolayers in a Langmuir trou gh . I t i s in t e r e s t in g to n ote th a t corn l e a f b a ses showed f a i r l y h igh r e l a t i v e a d so r p tio n in th e s e t e s t s . In view o f th e above d i f f i c u l t i e s , fu rth er work was co n fin ed to th e m ito ch o n d ria l f r a c t io n . i s shown in F igu re 1 . The enzyme dependency and th e e f f e c t o f fr e e z in g Each 0 ,1 ml o f m itochondria i s e q u iv a le n t to 0 .8 6 gms o f corn sh o o ts ( fr e s h w e ig h t). The in cu b a tio n m ixture volume was 2 .5 ml and i t co n ta in ed 12 juM sodium p y ru v a te, 2 .5 JiM sodium s u c c in a te , 0 .1 juM ATP, 5 juM MgClg, 2 .5 JtiM o f L -tryptophan and su cro se to make th e a ssa y is o to n ic . I t may be seen th a t 0 ,6 ml gave about th e maximum a c t i v i t y , and th a t fr e e z in g tw ic e red u ced th e a c t i v i t y g r e a t ly . The h ig h e st a c t i v i t y would be e q u iv a le n t t o about 50 /ig o f IAA, or about 10$ co n v ersio n in 8 hours a t 30°C. I t was ap p aren t, however, th a t o th e r Salkow ski r e a c t iv e p rod u cts were p rob ab ly p r e s e n t. For one th in g , th e r a t e o f c o lo r d evelop ­ ment was much f a s t e r than fo r s y n t h e t ic IAA added t o th e in cu b a tio n m ix tu res. Some in d o le -3 -p y r u v ic a c id was o b ta in ed through th e c o u r te sy o f Dr. H. S e l l , and t h i s sample a ls o gave v ery ra p id c o lo r developm ent w ith Salkow ski r e a g e n t. In a d d itio n , i f p h en ylh yd razin e h y d ro ch lo rid e was added to th e d e p r o te in iz e d in cu b a tio n m ixtures and allow ed to stan d 30 m in u tes, a s l i g h t t u r b id it y formed. A fte r removing th e t u r b id it y by c e n t r ifu g a t io n , th e su p ern atan t was a ssa y ed w ith Salkow ski a s sa y . Color developm ent in t h is -3 7 KLETT READING (NO. 540) o • o ro o fot o ftO o a\ © « o 00 o « H* o o . t- * ro o • i- * •P - ftO fto fto ( -* CJN 1—* 00 ro o 1CO H* 0Q O ftO -P> M L ENZYME o o 00 I—1 O Ml -3 8 c a s e was slow and more t y p ic a l o f IAA. When ph enylh ydrazine hydro­ c h lo r id e was added to a s o lu t io n o f 4 mg in d o lep y ru v ic a c id in 2 ml o f 50 $ e th a n o l, a c r y s t a lin e p r e c ip it a t e was o b ta in ed . The IAA formed from tryptophan by in cu b a tio n w ith corn m itochondria was i d e n t i f i e d by eth e r e x t r a c t io n , fo llo w e d by paper chromatography ( isop rop an ol/am m on ia/w ater, 8 : 1 :1 ) o f th e a c id eth er f r a c t io n . When la r g e amounts o f in c u b a tio n m ixture were chromatographed, s e v e r a l sp o ts appeared, w ith e it h e r E h r lic h or Salkow ski r e a g e n ts , in a d d itio n to th e IAA s p o t. In a l l c a s e s , th e IAA d e te c te d was much l e s s than estim ated from d ir e c t S alk ow sk i a s sa y . The o th er unknown in d o le compounds appear­ ed to correspond to th e breakdown prod ucts o f in d o lep y ru v ic a c id when i t was chrom atographed, and w i l l be co n sid ered below in con n ection w ith experim ents u sin g la b e le d DL-tryptophan as a s u b s tr a te . Other experim ents in t h i s la b o r a to r y had c a s t some doubt upon try p ­ tophan b ein g th e main p recu rso r o f IAA in e x c is e d corn s h o o ts . There­ f o r e , experim ents were d e v ise d to fin d i f IAA r e a l l y does come norm ally from tryptophan in th e p la n t. I t i s known th a t in Avena c o le o p t ile s t h e t i p i s th e s o le source o f auxin; and i f th e t i p i s removed, growth w i l l sto p c o m p le te ly , or a t l e a s t fo r an hour or so u n t i l th e r e i s so c a lle d r e g e n e r a tio n . Avena cu rvatu re t e s t . T his i s th e reason fo r double d e c a p ita tio n in th e I t was thought th a t th e t i p should have th e system fo r c o n v e rtin g tryptophan to IAA i f t h is i s th e major pathway, and th e b a sa l p o r tio n o f th e c o l e o p t i l e s would be un able to con vert any tryptophan to IAA, However, i t took about one hour to h a r v e st th e 320 c o l e o p t i l e s ; th us some o f th e b a sa l segments cou ld have acq u ired th e a b i l i t y t o co n v ert tryptophan t o IAA. ( s p e c i f i c a c t i v i t y - 0 .4 3 D L -tryptop han -2-C ^ was used so th a t th e r e would be no -3 9 q u e stio n about th e IAA produced coming from tryp top h an . Three hundred and tw en ty 3 -d a y -o ld o a t c o l e o p t i l e s (grown in th e dark a t 25 ° and 95$ r e l a t i v e hum idity) w ere d e c a p ita te d (5 nun from t i p ) . th e rem aining c o l e o p t i l e stumps were h a rv ested . 0*95 grams w h ile th e stumps weighed 6 ,2 grams. The t ip s a s w e ll as The w eig h t o f t i p s was The m itochondrial f r a c tio n s were i s o l a t e d from b o th , as p r e v io u s ly d e s c r ib e d , and resuspended in 1 ml o f i s o l a t i o n s o lu t io n . The enzyme c o n s is t e d o f 0 ,5 ml o f m itochondria and b o ile d enzyme c o n tr o ls were run. The a ssa y m ixture co n tain ed 12 juM sodium p y ru v a te , 2 .5 .uM sodium s u c c in a te , 0 .1 pM ATP, 5 -hM MgC^t 2 . 5 /jM D L-tryptophan and su c ro se to make th e a s sa y is o t o n ic in a volume o f 1 .7 5 m l. A fte r 8 hours o f in cu b a tio n a t 30°C, m ixtures were d e p r o te in iz e d w ith NH^SO^. The a c id f r a c t io n s were chromatographed (a scen d in g ) on paper (isop rop an ol/N H ^ /l^ O ). and rad ioau tograp h ed . The chromatogram was sprayed w ith E h rlich reagen t Both th e t i p s and th e c o l e o p t i l e s were a b le to con­ v e r t la b e le d tryptophan to la b e le d IAA, but th e t i p s were probably fa r more a c t i v e , k eep in g in mind t h a t th e fr e s h w eigh t o f th e stumps was about 6 .5 tim e s th a t o f th e t i p s . On rad ioautogram s, an in d o le d e r iv a t iv e was de­ t e c t e d j u s t below IAA in b oth th e b o ile d and u n b o iled e x t r a c t s . On paper i t gave an E h r lic h 1s r e a c t io n , and th e tryptophan sample was found t o con­ t a i n t h i s su b sta n ce as a contam inant. Another approach used was t o compare th e form ation o f IAA from oth er s u b s tr a te s w ith th e amount formed from tryp top han, u sin g th e corn homogenate. I t was th ou gh t th a t th e w hole homogenate should be used in th e s e e x p e r i­ ments b ecau se th e sup ernatan t might be a b le to con vert o th er s u b s tr a te s to IAA even though i t does n ot appear to con vert tryptophan to IAA. Though t h i s was t r i e d , no IAA could be d e te c te d , probably due to th e p r e v io u s ly d is c u s s e d d i f f i c u l t i e s . Another experim ent was t r i e d u sin g o n ly th e -4 0 m itoch on d ria and th e fo llo w in g s u b s tr a te s ; tryptop han , in d o le and s e r in e , in d o le and g l y c o l i c a c id , in d o le and g ly c in e , in d o le and a ce­ t a t e , in d o le and fr u c to s e -1 ,6 -d ip h o s p h a te , and in d o le a lo n e . Paper chromatography in d ic a te d t r a c e s o f IAA were formed w ith in d o le and s e r in e , in d o le and g ly c in e , and perhaps in d o le and g l y c o l i c a c id . How­ e v e r , no s u b s tr a te was as good as tryptoph an . The p o s s i b i l i t y e x is t e d th a t tryptophan was degraded to in d o le by th e m itochondria and th en in d o le and some 2 - carbon fragm ent such as g ly c o la t e was co n verted to IAA. The la b e le d IAA (form ed from tryptophan) would s t i l l have been p o s s i b l e , but th e s id e ch ain o f tryptophan would be ex p ected to be in co rp o ra ted in to many o th er com ponents, and some C ^ 0 2 m ight even be l o s t . T his i s b ased , o f c o u r se , on th e assum ption o f th e p resen ce o f a transammidase or an o x id a tiv e deam idation. In e it h e r c a s e , a r e a d ily m e ta b o liz a b le organ ic a c id would c o n ta in th e la b e l. The o v e r a ll r e a c tio n t o a la n in e and in d o le has been found in E. c o l i and i s c a l l e d tryp top h an ase (1 8 ? ). Of c o u r se , i f t h i s system was o p e r a tin g , one m ight ex p ect to fin d some in d o le on th e chromatograms. I t was p o s s ib le t o s e e i f th e m ito ch o n d ria l co n v ersio n was d ir e c t 14 or in d ir e c t by u sin g try p to p h a n -2 - C . A ll th e r a d io a c t iv it y should be in th e ca rb o x y l group o f IAA and th e s p e c i f i c a c t i v i t y o f IAA should be com parable to th a t o f tryp top h an. The d egrad ation o f IAA by therm al d eg ra d a tio n ( 1 2 9 ) should le a d to in a c t iv e s k a to le and -4 1 - The above experim ent was conducted fou r tim es w ith m itoch on d rial system s and tw ic e u sin g th e w hole corn s h o o ts . The f i r s t two experim ents were n o t c a r r ie d to com p letion due to th e sm all amount o f IAA ob ta in ed in th e f i r s t c a s e , and th e i n a b i l i t y to r e c r y s t a li z e to co n sta n t a c t i v i t y in th e second c a s e . The th ir d and fo u rth experim ents were com pleted. The w hole corn sh o o t experim ents f a i l e d due to th e sm all amount o f a c t i v i t y in th e IAA (700 cou n ts t o t a l ) i s o la t e d . C a rrier IAA had been added. In th e th ir d exp erim en t, 102 grams o f 4 -d a y -o ld corn sh o o ts were ground in 70 ml o f 0 .1 M phosphate 0 .4 M su c ro se b u ffe r pH 7*0 a t 1°C. The washed p a r t i c l e s (prepared as p r e v io u s ly d e sc r ib e d ) were resuspended in a t o t a l volume o f 12 ml in th e phosphate su cro se b u ff e r . T his i s a c o n c e n tr a tio n o f 0 .8 7 gms fr e s h w eigh t per ml o f m ito ch o n d ria l su sp en sio n . The in c u b a tio n m ixture used was 125 jM sodium p yru vate, 25 juM sodium 14 1 2 .5 pM M gC^, 25 pM ATP, 5*0 juM t r y p t o p h a n - 2 - C , a n d 6 .0 m l s u c c in a te , o f m ito c h o n d r ia i n a t o t a l a s s a y v o lu m e o f 25 m l. The in c u b a tio n p er io d was 8 hours a t 30 d egrees C. Salkow ski e stim a te s on th e su p ern atan t a t t h is tim e in d ic a te d an apparent IAA c o n c e n tr a tio n o f about 10 p g p er m l. The m ito ch o n d ria l p a r t ic le s were spun o f f , th e pH a d ju ste d t o 3 .0 w ith t a r t a r ic a c id and th e supernatant e x tr a c te d 3 tim es w ith e th e r . o f 57°C. The e th e r was evaporated o f f under w ith a bath tem perature The r e s id u e was p a r t it io n e d between n-hexane and a c e t o n i t r i l e and th e n-hexane d isc a r d e d . The a c e t o n i t r i l e was th en evaporated o f f as ab o v e, and th e r e s id u e tak en up in 10 drops o f EtOH, The a lc o h o l s o lu t io n was chromatographed as a 3 - in ch s t r i p on washed Whatman 1 paper (iso p r o p o n a l/ ammonia/water 8 ;1 :1 v / v ) . The paper was e q u ilib r a te d fo r 4 hours and run 16 hours a t 1°C. There was a sm all amount o f IAA p r e se n t as in d ic a te d by flu o r e s c e n c e -4 2 and jo-dim ethylam inobenzaldehyde (PDMABA) t e s t o f a | c e n te r o f th e 3 -in c h band. in ch s t r ip in th e The l a t t e r t e s t in d ic a te d 4 compounds were p r e s e n t in a d d itio n to IAA (T able V I). The re-chrom atography o f sp o ts A, C and D in d ic a te d ap p roxim ately th e same Rf v a lu e s . The IAA was removed from th e paper m oistened w ith 0.1M t a r t a r ic a cid pH 3 -0 by e x tr a c tio n 3 tim es w ith e th e r . The eth e r was co n cen tra ted , evap orated on a g la s s cou n tin g p la n c h e t, and i t counted 19,630 CPM. IAA was washed from th e p la n ch et w ith 2 ml a lc o h o l. evaporated under Ng The The a lc o h o l was 57°0 and a Salkow ski t e s t in d ic a te d 5 Pg o f IAA was p r e s e n t, or 10 jug t o t a l . The r a t io o f th e m olecu lar w eig h ts o f tryp top h an and IAA i s 0 .8 7 5 and th e r a t io o f th e cou n ts per 10 jug i s 0 .8 6 0 . (Under th e s e c o n d itio n s , 10 jug tryptophan = 16,850 cpm). The s p e c if i c a c t i v i t y o f th e IAA i s o la t e d was in good agreement wuth th e s p e c i f i c a c t i v i t y o f th e tryptophan fe d . The fo u rth experim ent was ca r r ie d to com p letion and both th e s p e c if i c a c t i v i t y o f th e IAA and th e p o s it io n o f th e la b e l would su g g est d ir e c t c o n v e r sio n o f tryptophan to IAA. V II. T his experim ent i s summarized in Table -4 3 TABLE V I R e s u lts o f In cu b a tin g T ryp toph an-2-C ^ w ith th e M itochondrial F r a c tio n from 5-Day-Old E t io la te d Corn S h o o ts. S ubstances D e te c te d on Paper Chromatograms o f th e A cid Ether E x tr a c t. Su b stan ce Approximate R a d io a c tiv ity Rf^ Color With: Salkow ski E h rlich UV L igh t Y ellow Pink F. L t. Blue A 0 .9 0 50 B 0 .5 8 640 Pink — — IAA 0 .4 3 1 1058 Red Purple F. L t . Blue C 0 .3 1 1 640 Red Pink F. L t. Blue D Q.241 846 Red Red F. L t. Blue ^Streaking in t h i s area o f chromatogram ^1sopropanol/am m onia/w ater ( 8 s l s l ) TABLE V II P u r if ic a t io n and D egradation o f R a d io a ctiv e IAA Formed From In cu b ation o f Corn Shoot M itochondria w ith T ryptop han-2-C ^ IAA-C1^ E lu ted + 50 me IAA R e c r y s ta liz a tio n ^ 1 IAA BaC03^ 2 1 7 .6 " » 2 18 7 .7 11 »i j 1 7 3 .4 ir 11 R e c r y s t a liz a t io n from 50$ eth a n o l ^ A fter one r e su b lim a tio n ^C orrected fo r th ic k n e s s cpM/10 m S katole^ 2 0 9 .2 0 .4 1 8 9 .? -M - I s o la t io n o f 3 - I n d o ly la c e t ic A cid from U s tila g o zeae Tumors ♦ by G. Turian I n s t i t u t e o f G eneral Botany, U n iv e r s ity o f Geneva (S w itzerla n d ) and R. H. Hamilton U n ited S t a te s Department o f A g r ic u ltu r e , A g r ic u ltu r a l R esearch S e r v ic e and Department o f Botany and P la n t P ath ology M ichigan S t a t e U n iv e r s it y , East L an sing, M ichigan (U .S .A .) Smut i n f e c t io n s cause an in c r e a s e in th e auxin con ten t o f th e h o st t is s u e s ^ . T h is was f i r s t dem onstrated in U s tila g o zeae tumors by 2 Moulton , u sin g th e Avena cu rvatu re t e s t . Wolf 3 rep o rted th a t fou r s t r a in s o f U. zeae produced au x in , i d e n t i f i e d as in d o ly a c e tic a c id (IAA) i n a medium c o n ta in in g tryp top h an . Von G uttenberg and S tu tz and H irata^ a ls o d e te c te d au xin by b io a s s a y in U. zeae c u ltu r e s and in th e corn tum ors. T u ria n 's o b serv a tio n ^ o f h igh c a t a la s e l e v e l s in U. zeae 7 tumors and th e a c t iv a t io n o f a c id ph osph atase by tumor e x t r a c ts , a ls o supported th e occu rren ce o f a h igh IAA l e v e l . However, upon chromatoo graphy o f an e x tr a c t from sm all q u a n t it ie s o f tum ors, no IAA could be d e te c te d w ith Salkow ski r e a g e n t. Turian th u s concluded th a t th e IAA c o n c e n tr a tio n ( i f p r e se n t a t a l l ) must have been low er than 10”^M. These r e s u l t s , to g e th e r w ith th e ab sence or a t th e most a v ery low co n c e n tr a tio n 9*10 o f IAA in h e a lth y corn s e e d lin g s 9 , su g g ested th a t chem ical d e t e c tio n o f th e p a t h o lo g ic a l hyperauxiny in th e tum orized t i s s u e s would r e q u ir e ♦Supported in p a rt by U. S. N a tio n a l S c ie n c e Foundation C o n trib u tio n No. 257^ from M ichigan A g r ic u ltu r a l Experiment S ta tio n -4 5 e x t r a c tio n o f much la r g e r amounts o f tumor than p r e v io u s ly u sed. S in ce th e low c o n c e n tr a tio n o f IAA and th e h igh c o n c e n tr a tio n o f in t e r f e r in g su b sta n ces p r ec lu d e d ir e c t ch em ical d e t e c tio n , i t i s n e c e ssa r y to u se m u lt i-s t e p p u r i f i c a t i o n p ro ced u res. These proced ures r e s u lt in g rea t l o s s o f IAA during p u r if ic a t io n ; so a sm all amount o f IAA-2-C-*-^ was added to p erm it c o r r e c tio n fo r t h e s e l o s s e s . The r a d io a c tiv e IAA i s a ls o ex trem ely u s e f u l in lo c a t in g and fo llo w in g th e IAA during th e is o la tio n . Three main experim ents were performed; th e f i r s t w ith 1500 gms o f m aturing tumors ( early-ch lam ydospore s t a g e ) , th e second w ith 1200 gms o f younger tumors (m ain ly pre-chlam ydospore s t a g e ) , and th e th ir d w ith 1840 gms o f u n in fe c te d corn s t a lk s ( e a r l y - t a s s e l s ta g e , c o lle c t e d in th e f i e l d a t th e same tim e as th e younger tumors -20°C u n t i l u se d . The m a te r ia ls were sto r e d a t The data and th e fo llo w in g t e c h n ic a l d e s c r ip tio n correspond to our i s o l a t i o n o f IAA from younger tum ors, and s e r v e s t o I l l u s t r a t e th e g en er a l methods u sed . Tumors were ground in a Waring blendor w ith s u f f i c i e n t 95$ e th y l a lc o h o l t o make th e f i n a l a lc o h o l c o n c e n tr a tio n about 80$. A fte r f i l t r a ­ t i o n , I . 6 5 jag o f IA A -2-C ^ ( s p e c i f i c a c t i v i t y o f 9 .1 8 x lQ^d/m/pg^-*- o r, under our co u n tin g c o n d itio n s , 34,500 c /m /p g ), 7 grams o f eth y len ed ia m in et e t r a a c e t i c a c id (EDTA), and s u f f i c i e n t NaHCO^ to make th e e th a n o lic e x t r a c t a lk a lin e were added. The eth a n o l was removed in vacuo a t 37°C by u se o f a stea m -d riv en "spray dry" ap p aratus, and th e r e s u lt in g 1 .5 l i t e r s o f e x tr a c t fu r th e r co n cen tra ted to about 700 ml w ith a r o t a t in g film evap orator a t 50°C and 17 mm Hg. The co n cen tra te (pH about 8 . 5 ) was f i l t e r e d through g la s s wool and e x tr a c te d w ith two volumes o f p e r o x id e f r e e d ie t h y l e th e r , and th e eth er was d isca rd ed . The aqueous p o r tio n -4 6 was a d ju ste d to pH 3->5 w ith 10 N e th e r • and e x tr a c te d w ith 3 volumes o f The combined e th er f r a c tio n s were con cen trated jLn vacuo to about 100 ml and e x tr a c te d tw ic e w ith 30 ml o f 8$ NaHC0 3 . The combined b ica rb o n a te e x tr a c ts were a d ju sted to pH 4 .0 w ith 10 N H^PO^ and ex­ tr a c te d 3 tim es w ith equal volumes o f e th e r . The combined eth er e x tr a c ts were d r ie d over anhydrous sodium s u lf a t e a t 1°C, and then con cen trated in vacuo to a dark reddish-brow n ’'o il" . This r e s id u e was d ilu te d to 20 ml w ith e th e r , and a 0 .2 ml a liq u o t was counted on an etched g la s s p la n c h e t (2 .6 9 sq cm area) by u sin g a thin-w indow gas flo w G.M. tu b e . The t o t a l r e a d io a c t iv it y in t h i s f r a c tio n was 36,320 cpm, or about 64$ o f th e IAA-2-C1^ added. The " o i l ” con tain ed c o n sid e r a b le a c id ic l ip o id a l m a te r ia l, p o s s ib ly h y d r o ly tic p rod u cts o f th e g lu c o lip id u s t i l a g i c a c id 1o . In an e f f o r t to se p a r a te IAA from t h i s l i p o i d a l m a te r ia l, th e " oil" was p a r titio n e d 2 tim es ( a f t e r removing th e e th e r ) between 5 ml o f a c e t o n i t r i l e and 5 ml o f lig r o in . The l i g r o i n la y e r s ( l i g h t y ello w ) were d iscard ed ( l e s s than 200 co u n ts; about 0 .2 $ o f th e IAA i s l o s t w ith each e x t r a c tio n ) . A fte r removal o f th e a c e t o n i t r i l e in v a cu o , column chromatography was attem pted by u sin g 25 gm o f diatom aceous s i l i c a (a n a ly tic a l-g r a d e C e lit e , JohnsM a n v ille C o ., U .S .A .) m oistened w ith 20 ml o f 0 .1 M phosphate pH 6 .5 0 as th e s t a t io n a r y p h a se, and e th er sa tu ra ted w ith th e same b u ffe r as th e m ob ile p h ase. 26 f r a c tio n s o f 3 .5 ml were c o lle c t e d and r a d io a c t iv it y was e stim a te d by cou n tin g 0 .2 ml a liq u o ts from each tu b e . F r a c tio n s 6 -1 8 were combined and d ried over anhydrous sodium su lp h a te , 'and th e e th e r was th en removed in vacu o. The r e sid u e s were paper chromato­ graphed fo r 14 hours on Whatman 3mm paper (p r e v io u s ly washed w ith a lk a lin e EDTA and 0 .1 N HCL) in isopropanol/am m onia/w ater ( 8 j 1 :1 ) as -4 7 th e a scen d in g s o lv e n t . The IAA runs ju s t above, but does not sep a ra te c o m p le tely from a dark-brown pigm ent; so t h i s e n t ir e area was e lu te d w ith 50$ e th a n o l. The e lu te d sample was rechromatographed as above on a 6 -in c h s t r i p but w ith a s l i g h t l y more aqueous s o lv e n t ( 7 ; 2 i l ) . Upon exam ination o f th e chromatogram under UV l i g h t and m on itoring fo r r a d i o a c t iv i t y , i t appeared th a t some sep a ra tio n o f IAA from th e pigment had been a ch iev ed so t h a t th ey cou ld be e lu te d s e p a r a te ly . A fte r e lu t io n and co u n tin g as above, th e dark-brown-pigm ented f r a c tio n con­ ta in e d 8 ,3 5 0 cpm, w h ile th e lig h t - y e llo w f r a c t io n had 22,780 cpm. a t t h i s s ta g e , t o t a l IAA reco v ery was about 55$* Thus S in c e 63 $ o f th e IAA was in th e li g h t - y e l l o w f r a c t io n , t h is was fu rth er p u r ifie d by paper e le c t r o p h o r e s is a t 1°C on Whatman 3 MM paper (washed as above) by u sin g 0 .1 M c i t r a t e b u ffe r o f pH 5.25* The m o b ility o f IAA under t h e s e con­ d it io n s as judged by th e a p p lic a tio n o f known IAA to one edge o f the electro p h o reto g ra m , i s 2 .9 4 x 10~^cirfV v o l t - s e c . A fte r d ry in g , th e IAA was lo c a te d by means o f a UV l i g h t , by r a d io a c t iv it y , and by sprayin g a narrow t e s t s t r ip c o n ta in in g known IAA w ith E h r lic h 's reagen t (1$ p-dim ethylam inobenzaldehyde p lu s 8 . 5$ HC1 in 95$ e th a n o l.) The pigm ent- f r e e IAA area was e lu te d w ith 50$ e th a n o l, and th e e lu t io n volume was a d ju ste d to 1 .5 m l. Measurements o f th e reco v ery o f r a d io a c t iv it y on d u p lic a te 0 . 0 5 ml sam ples in d ic a te d a recovery o f 10,7 3 7 cpm, or 1 8 .8 $ o f th e IAA-2-C^if' added. For th e q u a n tita tiv e e stim a tio n o f th e IAA re c o v e re d , a 0 .3 ml sample was assayed w ith 1 .2 ml o f Salkow ski reagen t 13 by u sin g th e K le tt m ic ro co lo rim eter w ith a No. 5^ f i l t e r . The r a te o f c o lo r developm ent, fo llo w e d a t 10-m inutes in t e r v a ls fo r 40 m in utes, was about th e same in both standards and sam ple. was 4 .0 jug per 0 =3 ml. The IAA co n cen tr a tio n Thus a t o t a l o f 20 jug o f IAA was is o la t e d . -4 8 S in c e about 0 .3 Mg o f t h i s IAA was th e IAA-2-C1^ added, th e t o t a l non­ r a d io a c t iv e IAA i s o l a t e d would be 19*7 Mg and th e t o t a l IAA p resen t in th e tumor t i s s u e was 105 M g. R e s u lts o f th e th r e e i s o l a t i o n s are summarized in th e fo llo w in g t a b le . The IAA Content o f U s tila g o zeae Tumors and Corn S ta lk s Based on I s o la t io n and C o lo rim etric D e te c tio n o f IAA and C orrected fo r Recovery o f Added IAA-2-C-^ Fresh Wt. P ercent jug IAA/Kilo o f Fresh jug IAA Recovery o f Weight C orrected For I s o la t e d 3, IAA-2-C1^ Recovery (M. C one.) T issu e (m s) Maturing tumors ( e a r ly chlamydospore s ta g e ) 1500 5-85 18.5 2 1 . 0 ( 1 . 2 x l c r 7) Younger tumors (m ain ly p r e chlam ydospore s ta g e ) 1200 19.70 18.8 8 7 .5 ( 5 . 0 X 10 1840 1.10 14.3 4 .2 ( 2 .4 x 10 - 8 ) ) Q Corn s t a lk s ( e a r ly t a s s e l ) a. C orrected fo r reco v ery o f IA A -2-C ^ . Amount a d d e d was fo r maturing tu m o r s 0 . 8 2 5 Mg» f o r y o u n g e r tu m o r s 1 . 6 5 Mg and f o r c o r n s t a l k s 3 - 3 0 Mg. b. A fte r column chromatography, h a lf o f th e combined IAA f r a c t io n was paper chromatographed in isopropanol/am m onia/w ater ( 8 s l s l ) , and nbutanol/am m onia/w ater ( l O s l s l ) s u c c e s s iv e ly . The e lu e n t from th e l a t t e r paper chromatogram was recombined w ith th e rem aining o n e -h a lf IAA f r a c t io n from th e column. The combined f r a c tio n s were again column-chrom atographed, paper-chromatographed a s above (iso p r o p a n o l/ am m onia/w ater), and e le c tr o p h o r e se d . c. A fte r column and paper chromatography (isop rop an ol/am m on ia/w ater), th e IA A -containing f r a c t io n was e le c tro p h o resed tw ic e . The IAA c o n c e n tr a tio n was estim a ted in t h is ca se on th e f in a l e le c tr o p h o r e to gram by sprayin g i t w ith E h r lic h ’ s reagen t and comparing i t w ith IAA sta n d a rd s. Recovery o f IAA-2-C-^ was estim a ted ju st b efo re th e f i n a l e le c tr o p h o r e s is on d u p lic a te a liq u o t s , and a radioautogram in d ic a te d th a t a l l th e r a d io a c t iv it y on th e f i n a l electrop horetogram was IAA, -4 9 - The IAA c o n c e n tr a tio n s have been determ ined in U s tila g o zeae tumors in two s ta g e s o f developm ent, and in v e g e t a tiv e corn s t a lk s ( e a r l y - t a s s e l sta g e ). The v a lu e s should be co n sid ered rough ly q u a n t it a t iv e , b ein g lim it e d by th e c o lo r im e tr ic methods u sed . I t should be noted th a t th e p a r t it i o n column was not a s a t is f a c t o r y p u r if ic a t io n ste p in t h is stu d y , though i t has been employed s a t i s f a c t o r i l y in th e c a se o f corn s e e d lin g s . 9 I t appeared th a t th e proper p a r t it io n was p reven ted by th e r e s id u a l o i l ­ l i k e r e s id u e o b ta in ed upon c o n cen tra tin g th e a c id eth e r f r a c t io n s . The o n ly comparable r e s u lt s from a q u a n tit a tiv e b io a ssa y are th o se o f Moulton . He f a i l e d , however, to f in d p r o p o r t io n a lit y between th e amount o f tumor e x tr a c te d and th e cu rvatu re in th e Avena curvature t e s t , and su g g e sted th e p resen ce o f an in h ib it o r . He a ls o found c o n tin u a l pro­ d u c tio n or r e le a s e o f auxing fo r s e v e r a l days by c o ld -e th e r or w ater e x t r a c tio n s o f f r e s h or ly o p h y liz e d tumor. In one experim ent, in which he used w ater e x tr a c tio n a t 2°C fo r 1 hour, he found a cu rvatu re e q u iv a le n t to a p p roxim ately 15-30 -Ug of IAA/gm fr e s h w eig h t. T his huge d iscrep an cy between our v a lu e s , based on i s o l a t i o n and d e t e c tio n , and M oulton's v a lu e s , based on b io a ss a y , may su g g e st th e p resen ce o f a d d itio n a l d i f f u s i b l e 4 a u x in s. However, von G uttenberg and S tr u tz rep o rt th a t th e auxin a c t i v i t y in an a lc o h o lic e x tr a c t o f tumor i s in a c tiv a te d by crude pea IAA o x id a se . In any c a s e , young U s tila g o zeae tumors a re c h a r a c te r iz e d by an IAA l e v e l much above th a t o f th e u n in fe c te d s t a lk t i s s u e . We w ish t o thank Dr. S . A. Gordon, who k in d ly su p p lied th e IAA-2-C and Dr. R. S. Bandurski f o r h is encouragement and s u g g e s tio n s . 14 , -5 0 L ite r a tu r e C ited 1. H. E. Gruen, Ann. Rev. P la n t P h y s io l. , 10 (1959) ^05 2. J . E. M oulton, Botan. G a z ., 103 ( 19 ^2 ) 725 3. F. T. W olf, P roc. N a tl. Acad. S c i. U .S ., 38 (1952) 106 H.von G uttenberg e t I . S tr u tz , Arch. M ik r o b io l., 17 (1952) 189 5* S . H ir a ta , Ann. P h y to p a th o l. Soc, Japan, 22 (195?) 153 6. G. T urian, Rev. Gen. B o ta n ., 65 (1958) 279 7. G. T urian, E x p e r ie n tia , 13 (195?) 368 8. G. T urian, Arch. S c i. Geneva, 9 (1956) ^65 9. R. H. H am ilton, unpubl. r e s u lt s 10 . S. H ou sley, A. Booth e t D. J. P h i l l i p s , N ature, 1?8 (1956) 255 11. R. E. S tu tz , D. E. A tk in son , and S. A. Gordon, Argon. N a tio n a l L a b o ra to ries R eport No. A-?10 (1951) 12 . R. U. Lemieux, Can. J. Chem., 31 (1953) 396 13. Y. W. Tang e t J. Bonner, Arch. B ioch em ., 13 (19^7) 11 -5 1 S tu d ie s on I s o la t io n o f I n d o le -3 -A c e tic A cid From Corn K ernels and E tio la te d Corn S eed lin g s R. H, Hamilton Crops R esearch D iv is io n , A g r ic u ltu r a l Research S e r v ic e U n ited S t a te s Department o f A g r ic u ltu r e and R. S . Bandurski and B. H. G rigsby Department o f Botany and P la n t P ath ology M ichigan S t a te U n iv e r s ity E ast L ansing, M ichigan INTRODUCTION I n d o le - 3 - a c e t ic a c id (IAA) has been commonly accep ted a s an im p ortan t, i f n ot th e p r in c ip a l, p la n t auxin ( 5 .1 5 » 2 l) . Grain o f corn (Zea mays) i s a r ic h sou rce and m illigram q u a n tit ie s have been i s o la t e d by a lk a lin e h y d r o ly sis o f th e mature k er n e ls ( 3 * 1 7 )i or by d ir e c t e x tr a c t­ io n from immature k e r n e ls ( 1 6 ). A la r g e number o f r ep o rts have a ls o appear­ ed con cern in g th e o ccu rren ce o f IAA in v e g e t a t iv e p o r tio n s o f p la n ts ; but in many c a s e s d ie t h y l eth e r has been used as th e e x tr a c tin g s o lv e n t in a manner w hich can a llo w th e enzym atic co n v ersio n o f tryptophan to IAA ( 4 3 ). In a g r ea t many o th er c a s e s , th e e x is te n c e o f IAA i s presumed from th e b io a ssa y o f chromatograms run in a s in g le s o lv e n t . Thus p ro o f th a t f r e e IAA i s g e n e r a lly d is t r ib u t e d in growing p la n t t i s s u e i s not y e t a v a ila b le ( 2 ). In s tu d ie s rep o rted elsew h ere ( 3 8 ), r e l a t i v e l y la r g e amounts o f in d o le - 3 - a c e t ic a c id (IAA) have been d e te c te d in U. Zeae tumors, and sm a lle r amounts in h e a lth y , e a r ly - t a s s e l - s t a g e corn s t a lk s . The tec h n iq u es h ere rep orted perm it hand ling o f la r g e amounts o f t i s s u e and q u a n t it a t iv e e v a lu a tio n o f lo s s e s during i s o l a t i o n . They are -5 2 d esig n ed to measure f r e e IAA (th a t i s , IAA p r e se n t when th e t i s s u e i s q u ic k ly k i l l e d by hom ogenization in eth a n o l) and to perm it p h y s ic a l c h a r a c te r iz a tio n o f th e IAA so e x tr a c te d . U sing t h i s method, i t was not p o s s ib le to i s o l a t e any f r e e IAA from 80$ eth an ol e x tr a c ts o f corn seed ­ l i n g s i f th e pH o f th e aqueous c o n cen tra te was a c i d i f i e d to pH 4 .0 and e x tr a c te d w ith e th e r . Recovery o f IAA added to f i l t e r e d eth an ol e x tr a c ts or eth a n o l homogenates from kilogram q u a n t it ie s o f corn sh o o ts i s d is ­ cu ssed . I t i s su g g e sted th a t IAA may complex w ith eth an ol s o lu b le sub­ s ta n c e s from th e corn s h o o ts , and th e p ercen tage l o s s i s g r e a te r w ith 2 -3 Mg than w ith 100 jug. By a d ju s tin g th e aqueous co n cen tra te to pH 8 .0 w ith sodium b ica rb o n a te p r io r to eth er e x tr a c tio n , th e reco v ery o f 2 -3 MS IAA-2-C 14 was somewhat in crea sed and IAA could then be d e te c te d in e t i o la t e d 5 -d a y -o ld corn s h o o ts . P o s s ib ly , th e r e fo r e , IAA in viv o e x i s t s a s a complex th a t i s v ery e a s i l y hydrolyzed by m ild a l k a l i . Free IAA could be is o la t e d from corn k e r n e ls , but much more could be r e le a s e d fo llo w in g a lk a lin e h y d r o ly s is o f a gummy, y ello w -o ra n g e p r e c i p i t a t e r e s u lt in g from c o n cen tra tio n o f th e 80$ eth an ol e x t r a c ts . This p recu rso r appeared id e n t ic a l to th a t rep orted by Berger and Avery ( 4 ) , and may be an a lc o h o l s o lu b le p r o te in . METHODS M ichigan 350 hybrid corn was soaked in water fo r 4 hours and germin­ a ted on wet ab sorbent paper in p l a s t i c tr a y s fo r 5 days in th e dark a t 25°C and 90$ r e l a t i v e hum idity. The sh o o ts were h a rv ested by c u ttin g o f f th e c o l e o p t i l e p lu s th e f i r s t in ter n o d e. They were im m ediately homogenized in a blendor w ith s u f f i c i e n t 95$ e th y l a lc o h o l to make th e f i n a l c o n c e n tr a tio n 80$ eth a n o l. A fte r 1 -4 hou rs, th e eth an ol homogenate was f i l t e r e d and th e r e s id u e r e -e x tr a c te d once or tw ic e w ith 80$ e th a n o l. -5 3 The combined e th a n o lic e x t r a c ts were co n cen tra ted a t 55°C in vacuo w ith a r o t a t in g evap orator or in l a t e r experim ents w ith a vacuum spray d r ie r a t 35°C. In experim ents where IAA-2 - C ^ was added to perm it recovery c a lc u la t io n , i t was added to th e eth a n o l f i l t r a t e or t o th e homogenate p r io r to f i l t r a t i o n , w ith id e n t i c a l r e s u lt s . The aqueous co n cen tra te was a d ju ste d to pH 4 .0 w ith 5N HgSO^ or H^PO^ and th e p r e c ip it a t e r e ­ moved by f i l t r a t i o n through g la s s w ool. In l a t e r i s o l a t i o n s (where noted in t e x t ) , th e pH o f th e aqueous co n cen tra te was a d ju sted to 8 ,0 w ith NaHCO^. The aqueous co n cen tra te was e x tr a c te d 3 tim es w ith perox­ i d e - f r e e d ie th y le t h e r (shaken w ith s o li d FeSO/4, b efo re d i s t i l l a t i o n ) . In th e former ca se ( e x t r a c t io n pH 4 . 0 ) , th e eth er was p a r t it io n e d w ith 8$ NaHCO^ 3 t im e s , th e combined b ica rb o n a te f r a c t io n a c i d i f i e d to pH 4 .0 (5N ^SO/j,) and e x tr a c te d in to eth e r 3 tim e s. In th e l a t t e r ca se (ex ­ t r a c t io n pH 8 . 0 ) , th e e th e r was shaken once w ith 8 $ NaHCO^. A fte r a c i d i ­ f i c a t i o n o f combined aqueous b ica rb o n a te f r a c t io n s (a s a b o v e ), th e y were e x tr a c te d w ith eth e r th r e e tim e s . The combined eth er f r a c t io n was again b ica rb o n a te p a r t it io n e d as d escrib e d above. The a c id eth er f r a c tio n s were d rie d over anhydrous Na^SO^ a t 1°C and th e eth er removed in vacu o. The r e s id u e was washed 3 tim es w ith 5 i'll o f eth er and th e combined eth er f r a c t io n s reduced in volume to about 3 ml* This f r a c t io n was a p p lie d to a b u ffe r e d e th e r-w a ter p a r t it io n column ( s e e below) and e lu te d w ith e th e r . The IAA c o n ta in in g f r a c t io n s were p ooled and d ried over anhydrous sodium su lp h a te a t 1°C* The dry eth er was evaporated and th e r e s id u e was taken up in a few drops o f eth a n o l fo r chromatography or e le c t r o p h o r e s is . F r a c tio n s o f s u f f i c i e n t p u r ity cou ld then be fu r th e r fr a c tio n a te d by two d im en sio n a l electro p h o resis-ch ro m a to g ra p h y . In t h is procedure, th e -5 4 sample was s p o tte d toward one corner o f a T-shaped p ie c e o f b u ffe r sa tu r a te d p ap er. The f la p ( o f th e same le n g th and w idth as th e e le c t r o ­ p h o r e s is bed) was fo ld e d over and in s u la te d by a p l a s t i c s h e e t. A fte r e le c t r o p h o r e s is , th e paper was d ried and used fo r chromatography a t r ig h t a n g le s to th e d ir e c t io n o f e le c tr o p h o r e tic m ig r a tio n . Paper e le c t r o p h o r e s is was conducted in a c lo s e d s t r i p , s o li d sup port, p l e x i ­ g la s s chamber u sin g Whatman 3MM paper, 0 .1 M c i t r a t e b u ff e r , pH 5-25» 200 or 250 v o l t s , and a tem perature o f 1°C. In experim ents in v o lv in g more than a few hundred grams o f corn s h o o ts , th e elctro p h o resis-ch rom atograp h y procedure cou ld not be u sed, as th e IA A -con tain in g column f r a c tio n s were to o impure to be a p p lied as a sp ot to th e b u ffe r sa tu r a te d paper. Under th e s e c o n d itio n s , con tin uous flo w e le c t r o p h o r e s is proved to be v a lu a b le . The con tinu ou s flow e le c t r o ­ p h o r e s is u n it was c o n str u c te d a f t e r th a t o f Durrum (11) ex cep t th a t th e e le c tr o d e compartment was b a f f le d , and carbon e le c tr o d e s were u sed. Whatman 3MM paper was washed s u c c e s s iv e ly w ith eth y le n e d ia m in e te tr a a c e t a t e a t pH 8 ,0 , 0 .1 N HC1, and d i s t i l l e d w ater. E le c tr o p h o r e s is a t pH 6 .5 5 w ith 0 .0 2 5 M phosphate b u ffe r a t room tem perature ( in th e dark) was found to be s a t i s f a c t o r y . Under t h e s e c o n d itio n s , th e IAA m igrated h a l f way toward th e p o s i t i v e e le c tr o d e when th e wick was one in ch from th e n e g a tiv e edge, w h ile th e pigm ents d id n ot m ig ra te. A lthough paper e le c t r o p h o r e s is r e s u lte d in th e b e st sep a ra tio n o f IAA from th e a c id ic p igm en ts, paper chromatography was somewhat more con­ v e n ie n t and was used u n le s s o th e r w ise s ta te d . Whatman 3MM or Whatman 1 paper was used fo r a scen d in g chromatography u sin g iso p ro p a n o l, ammonium h y d ro x id e, w ater s o lv e n t ( 8 : 1 : 1 , v /v ) in most c a s e s . The sample was u s u a lly a p p lie d as a 5 cm band w ith known IAA sp o tte d a t one end so as -5 5 to o v erla p th e unknown s l i g h t l y . Chromatography was conducted in th e dark a t 1°C fo r 20 to 30 h o u rs, a f t e r e q u ilib r a tio n fo r 8 to 12 hours. IAA was lo c a te d on th e paper by a l ig h t - b lu e flu o r e sc e n c e under sh o r t­ wave, u l t r a - v i o l e t r a d ia tio n (2537 A) or by sprayin g (w ith E h r lic h 's re a g e n t) a s t r i p cu t from th e edge o f th e band c o n ta in in g th e c o -s p o tte d IAA. The E h r lic h ’ s rea g en t con tain ed 1 gm £-dim ethylam inobenzaldehyde d is s o lv e d in 91 • 2 ml o f 95$ e th a n o l and 8 .8 ml o f con cen trated HC1. Chromatograms were e lu te d w ith 50$ e th a n o l, and Salkow ski reagen t (35) was used fo r th e d e te c tio n o f e lu te d IAA. The red c o lo r complex was measured w ith a c o lo r im e te r u sin g a w ide band, 540 mp f i l t e r ; standard c o n c e n tr a tio n o f IAA were in clu d ed in each d eterm in a tio n . The m od ified Avena s e c tio n s t r a ig h t growth t e s t o f McRae e t a l . (25) was used fo r th e b io a s s a y s . The IAA-2-C1^ used in th e se s tu d ie s had a s p e c if i c a c t i v i t y o f 9 .1 8 x lO^d/m/pg ( 3 4 ). Due to p o s s i b i l i t y o f ra d io -d ec o m p o sitio n , th e sample was p u r if ie d by column chromatography and paper chromatography tw ic e during th e cou rse o f t h e s e in v e s t ig a t io n s . The sto ck s o lu t io n was kept as a v ery d ilu t e s o lu t io n in e th a n o l and sto r e d a t -20°C . A fte r th e i n i t i a l p u r i f i c a t i o n , no decom position products were noted when th e IAA-2-C-^ was chromatographed. Sugar b e e t s , green peas and cucumbers were f i e l d grown on th e Michigan S t a te U n iv e r s ity farm and h a rv ested im m ediately b efo r e u s e . P o ta to e s and bananas and sw eet corn were purchased l o c a l l y , w h ile e t io la t e d pea sh oots (A la sk a ) were grown fo r 6 days a t 25°C and 90$ r e l a t i v e hum idity. As c a r r ie r IA A -2-C ^ was not used in most o f th e s e i s o l a t i o n s , no reco v ery e s tim a te s were p o s s ib le . In some o f t h e s e i s o l a t i o n s , as n oted , i n i t i a l a lk a lin e eth e r e x tr a c tio n was u sed . -5 6 EXPERIMENTAL In e a r ly exp erim en ts, i t became apparent th a t corn s e e d lin g s con­ ta in e d h ig h c o n c e n tr a tio n s o f a yellow ish -b row n p h e n o l-lik e pigm ents5*'. Chromatograms o f corn s e e d lin g e x tr a c ts were v ery co lo r e d and i t was im p o ssib le to d e te c t jag amounts o f IAA by flu o r e s c e n c e under u l t r a - v i o l e t r a d ia tio n , or by E h rlich or Salkow ski r e a g e n ts. These d i f f i c u l t i e s have a ls o hampered th e i s o l a t i o n o f IAA by workers u sin g oth er p la n t m a te r ia ls ( 1 ,8 , 2 7 ,4 0 ) . Column chromatography was used in th e i s o l a t i o n o f IAA by K8gl e t a l . ( 2 2 ), and o th er columns have been d escrib ed by L in ser (24) and F is c h e r and Behrens ( 1 2 ). However, none o f th o se columns appeared to be e n t i r e l y s a t i s f a c t o r y under our p a r tic u la r c o n d itio n s . The s e v e r a l columns t r i e d in t h is stu dy were c e l l u l o s e , a sty r e n e ty p e polyam ine r e s in (Dowex AG 3 -X 4 ), n e u tr a l alumina (Woelm), N,Nd ie th y la m in o e th y lc e llu lo s e , and w a te r -e th e r p a r t it io n u sin g a n a ly t ic a l grade diatom aceous s i l i c a ( C e l i t e , Joh ns-M anville) as th e in e r t b a se. The c e l l u l o s e column channeled even w ith c a r e fu l packing w h ile w et. The n e u tr a l alumina and th e Dowex AG 3-^4 appeared t o bind th e IAA very t i g h t l y and i t cou ld not be r e a d ily e lu te d . made a v er y s a t i s f a c t o r y column. N ,N -d ie th y la m in o e th y lc e llu lo s e The IAA was adsorbed on th e column from 0 .0 0 5 N phosphate b u ffe r a t pH ? o0 and could be e lu te d by in c r e a s in g th e s a l t or b u ffe r c o n c e n tr a tio n . The column chosen fo r u se was a tr u e p a r t it i o n column em ploying a b u ffered aqueous phase adsorbed on C e lit e *The p r in c ip a l pigm ent was c o lle c t e d as a p r e c ip it a t e a f t e r c o n cen tra tio n o f th e a c id e th e r f r a c t io n p r io r to column chromatography. T his su b stan ce was r e c r y s t a liz e d from a c e t o n e - lig r o in y ie ld in g l i g h t brow nish-pink to w h ite m on o clin ic sw eet t a s t in g c r y s t a ls (C^HqO^N) m eltin g a t 160-161 C ( d e c .) . The i d e n t i f i c a t i o n and c h a r a c te r iz a tio n o f t h is sub stan ce w i l l be rep o rted elsew h ere. -5 7 and e th e r as th e m ob ile p h ase. This p a r t it io n was s im ila r to th a t used by H o lle y e t . a l . (19) ex cep t th e s e workers used a Craig ap paratus. U sing t h i s ty p e o f column, th e IAA could be e lu te d in a sm all volume o f eth e r and e a s i l y c o n ce n tr a ted . A n a ly tic a l grade C e lit e was th orou gh ly ground in a mortar w ith 0 .8 tim es i t s w eigh t o f phosphate b u ffe r . P o r tio n s o f th e damp C e lit e were s lu r r ie d in th e column w ith b u ffe r sa tu r a te d e th er and th e s lu r r y packed w ith a g la s s rod. A flo w r a te o f 0 .5 to 2 .0 ml p er m inute was ob tain ed w ith 3 to 5 pounds p r e ssu re per square in ch o f n itr o g e n . The column was e lu te d w ith eth er sa tu ra ted w ith th e same b u ffe r used on th e column. F igu res 1 and 2 in d ic a te th e e f f e c t o f m o la r ity and pH o f th e phosphate b u ffe r on th e e lu t io n o f IAA from a 2 .5 cm column c o n ta in in g 10 g o f C e lit e . High b u ffe r co n c e n tr a tio n s d ep ress th e s o l u b i l i t y o f IAA in th e aqueous p h ase. Over th e sm all range o f hydrogen io n c o n c e n tr a tio n s t e s t e d , th e r e was an alm ost lin e a r r e la t io n ­ sh ip between pH and r a te o f movement o f IAA through th e column. Maximum se p a r a tio n o f IAA and th e corn pigm ents was ob tain ed w ith 0.1M phosphate b u ffe r a t pH 6.5* Under th e s e c o n d itio n s , w ith a 1 .8 cm d ia m eter, 20 g column, IAA came o f f in th e 40 to 60 ml f r a c tio n s ; w ith a 2 .5 cm, 30 g column, th e IAA came o f f in th e 50 to 70 ml f r a c tio n s ; and w ith a 5 cm, 100 g column, th e IAA came o f f in th e 200 to 500 ml f r a c t io n s . The h ig h e s t c o n c e n tr a tio n o f IAA was found j u s t a f t e r th e f i r s t tube or two c o n ta in in g i t , in d ic a tin g th a t th e IAA band t r a i le d upward on th e column. U su a lly 2 -3 ml o f e th er were c o lle c t e d per tu b e , and th e IAA lo c a t e d by adding 4 ml o f Salkow ski rea g en t and sh ak in g. The c o lo r d evelop ­ ment i s somewhat slo w er than in th e reg u la r Salkow ski a s sa y . The pK o f IAA i s 4 .6 5 (23) and a t pH 6.5* IAA i s about 10$ un­ d i s s o c i a t e d , th a t i s , th e r a t io o f d is s o c ia t e d to u n d is s o c ia te d m o lecu les -5 8 - i s about 9 .0 . The u n d is s o c ia te d m olecu les t h e o r e t i c a l l y en ter th e flo w in g -e th e r p h a se, move some f i n i t e d is ta n c e down th e column, and th en d i s s o c i a t e a t an aqueous-phase boundary. T h is c y c le corresponds t o a t h e o r e t i c a l p l a t e a s d e fin e d in f r a c t io n a l d i s t i l l a t i o n column th e o r y . M artin and Synge (26) have shown th a t movement o f s o lu t e on a p a r t it io n column p er volume (A V) o f s o lv e n t p a ssed , i s d e fin e d by a b in om ial ex­ p a n sio n . I f th e c r o ss s e c t io n a l areas o f th e m obile phase Am, s ta tio n a r y phase As , th e ex cu rsio n v a lu e R ( s o lu t e m ovem ent/solvent movement) and th e t o t a l a r e a , A, a re known, then th e p a r t it io n c o e f f i c i e n t , may be c a lc u la te d from th e form ula: . _ A Am s^ - aT U sing a 20 g , 1 .8 cm diam eter column w ith 0 .1 M phosphate b u ffe r a t pH 6.5» th e v a lu e fo r R was e stim a ted to be 0 .2 2 7 and th e valu e fo r was 9 .8 . RESULTS I s o l a t io n and Recovery Experim ents Employing U nlabeled C a rrier IAA R ecovery experim ents are e s s e n t i a l i f e stim a te s o f th e amount o f IAA p r e se n t in a t i s s u e a re to be made. One hundred jug o f IAA was added to 1 l i t e r o f 95$ eth a n o l and c a r r ie d through c o n c e n tr a tio n , eth er e x t r a c tio n , column and paper chromatography. The IAA was lo c a t e d on th e paper chromato­ gram by UV flu o r e s c e n c e , and e lu te d w ith 50$ e th a n o l. The eth a n o l was removed in vacuo a t 5 5 °0 , 2 ml o f w ater added, and th e amount o f IAA estim a te d by Salkow ski a s s a y . T h is experim ent was rep ea ted tw ic e w ith o v e r a ll r e c o v e r ie s o f 50 and 58 $ . Other s im ila r exp erim en ts, where l o s s e s f o r in d iv id u a l ste p s were e v a lu a te d , in d ic a te d l o s s e s were sm all fo r each o p e r a tio n , but cu m u lative. -5 9 - The i s o l a t i o n o f IAA from 5 -d a y -o ld corn sh o o ts was n ext i n v e s t i ­ g a te d . E ig h t experim ents w ith o u t added IAA were conducted u sin g from 178 g to 1000 g o f s h o o ts . m ents. No IAA cou ld be d e te c te d in th e s e e x p e r i­ Three rec o v e ry experim ents w ith known amounts o f added IAA were n ex t conducted. In th e f i r s t experim ent, 100 jig o f IAA was added to th e 80$ eth a n o l e x tr a c t o f 1000 g o f corn s h o o ts . S in c e some l o s s m ight be ex p ected owing to a d so r p tio n by th e r e s id u e , another 100 jag o f IAA was mixed w ith th e r e s id u e and th e r e s id u e washed again w ith 2 l i t e r s o f 80$ e th a n o l. R ecovery in t h i s experim ent was 25 $ in both c a s e s . In th e second exp erim en t, 120 jag o f IAA was added to 318 g o f 5 -d a y -o ld corn sh o o ts b e fo r e hom ogenization in a b len d or. th e added IAA was reco v ere d . In t h is experim ent, 27$ o f In th e t h ir d experim ent, 2 0 0 j i g o f IAA was added to 700 g o f corn sh o o ts and th e r e c o v ery in t h is experim ent was 30$. I t th u s appears th a t reco v ery fo r th e o v e r a ll is o l a t i o n procedure u sin g 100 jug p er kilogram o f corn sh o o ts i s 25 t o 30 $ . An attem pt was made to i s o l a t e IAA from 1 1 .3 kilogram o f 5 -d a y -o ld e t i o l a t e d corn s h o o ts . day in t e r v a l. F iv e l o t s o f corn sh oots were grown over a 30- Each in d iv id u a l l o t was h a r v e ste d , e x tr a c te d w ith 80$ e th a n o l, and worked up to th e p a r t it io n column s ta g e . T his a c id eth er f r a c t io n was co n cen tra ted to dryness in vacuo and sto r e d a t -20°C . The p o o led l o t s were d is s o lv e d in e th e r and th e eth er c o n cen tra te d iv id ed in to 3 p o r t io n s . Each p o r tio n was p a r titio n e d on a 5 cm d iam eter, 100 g column, and th e IAA f r a c t io n s p o o led . The p ooled elu a n t was e le c t r o ­ p h oresed in two runs u sin g th e con tin u ou s flo w u n it . d e te c te d by Salkow ski a s sa y . No IAA cou ld be When 26 jag o f known IAA were p la ced on th e 5 cm d iam eter column, reco v ery was 77$; and when 10 jag o f IAA was run on con tin u ou s flo w e le c t r o p h o r e s is , reco v ery was 70 $ . - 60 - Isolation experiments using IAA-2-C-^ S in c e th e above reco v ery experim ents might not be v a lid due to th e a d d itio n o f r e l a t i v e l y la r g e amounts o f IAA, low l e v e l reco v ery experim ents u sin g IAA-2-C-^ were c a r r ie d o u t. The u se o f t r a c e amounts o f h igh a c t i v i t y IA A -2-C ^ was v a lu a b le in s e v e r a l ways. Recovery data cou ld be ob ta in ed during th e a c tu a l i s o l a t i o n experim ents; 1 -3 jug could be added and d e te c te d by r a d io a c t iv it y , fo llo w in g i s o l a t i o n , even i f l o s s e s were 90$; and th e IAA cou ld be fo llo w e d during i s o l a t i o n by i t s r a d io a c t iv it y . The r e s u lt s o f th e s e is o l a t i o n experim ents are summarized in Table I . Corn g ra in was a r ic h source o f f r e e and bound IAA in con firm ation o f numerous r e p o r ts o f th e occu rren ce o f IAA in th e grain (3 » 8 ,1 6 ,3 2 ,3 3 * 4 0 ,4 4 ) . The grain was soaked 4 hours in tap water and allow ed to germ inate in th e dark a t 25°C and 95$ r e l a t i v e hum idity fo r 0, 12 or 24 hours. The samples were ground in a meat g rin d er, then ex tr a c te d 3 tim es w ith 1 to 3 l i t e r p o r tio n s o f 80$ e th a n o l. A gummy, yellow -oran ge p r e c ip it a t e was o b ta in ed upon c o n c e n tr a tio n o f th e eth a n o l e x t r a c ts . F ollow in g washing w ith w a ter, th e r e s id u e was hydrolyzed s u c c e s s iv e ly w ith two 150 ml p o r tio n s o f 1 N KOH fo r 1 hour a t 100°C. The h y d ro ly sa te s were th en c a r r ie d through th e d e scr ib e d IAA i s o la t io n procedure. The r e s u lt s are summarized in Table I I . The f r e e IAA and IAA r e le a s e d by h y d r o ly sis were c h a r a c te r iz e d by e le c t r o p h o r e s is , and by paper chromatography in 4 s o lv e n t s . The Rf v a lu e s o f th e s y n th e tic and is o la t e d IAA were id e n t ic a l in a l l s o lv e n ts (T ab le I I I ) , and th e r e was no sep a ra tio n o f th e c o -sp o tte d n a tu ra l and s y n t h e t ic IAA. The e stim a te o f c o n c e n tr a tio n , ob tain ed by th e Salkow ski a s s a y , was v e r i f i e d by d ir e c t sp ectrop h otom etric a ssa y o f chromatograms -6 1 - sprayed w ith E h r lic h ’ s r e a g e n t, and by Avena s e c t io n b io a ssa y (T ables IV and V ). The immature sw eet corn k e r n e ls con tain ed so much IAA th a t i f 1 g was ground w ith 1 .6 ml o f w ater, th e d e p r o te in iz e d s o lu t io n gave a stro n g Salkow ski t e s t . The o r ig in o f th e IAA r e le a s e d upon h y d r o ly sis i s o f i n t e r e s t . Other workers (1 3 ,2 9 » 4 0 ) have su g g ested th a t IAA may a r is e from p r o te in s c o n ta in in g tryp top h an . a lc o h o l s o lu b le p r o te in . Though z e in i s low in tryptop han , i t i s th e major A sample o f pure z e in A was hydrolyzed under th e c o n d itio n s used fo r th e corn grain r e s id u e , but no IAA was found. B erger and Avery (4 ) a ls o rep orted th a t t h e ir p recu rsor was not z e in . S im ila r a lk a lin e h y d r o ly sis o f 100 mg o f tryptophan i t s e l f y ie ld e d no IAA. To fu r th e r determ ine w hether th e p recu rsor was p r o t e in , about 500 g o f corn meal was e x tr a c te d th r e e tim es w ith 1 l i t e r p o r tio n s o f 0 ,0 1 M MgC^. The combined e x tr a c ts were co o led to 1°C and th e p r o te in p r e c ip i­ t a te d by s a tu r a tio n w ith ammonium s u l f a t e . The p r e c ip ita t e d p r o te in was c o l l e c t e d by f i l t r a t i o n and washed w ith 80$ e th a n o l. moved in vacuo a t 60°C. The eth a n o l was r e ­ The eth an ol s o lu b le and eth an ol in s o lu b le pro­ t e i n s were th en hydrolyzed fo r 1 hour in 1 N K0H a t 100°C. No IAA was found in th e h y d r o ly sa te o f th e eth an ol in s o lu b le p r o t e in , but a co n sid ­ e r a b le amount was o b ta in ed from th e eth an ol s o lu b le f r a c t io n . A number o f o th er p la n t t i s s u e s have been examined q u a l it a t iv e l y f o r th e p resen ce o f IAA and oth er E h rlich r e a c t iv e su b stan ces (Table V I). I t was in t e r e s t in g th a t v e g e t a tiv e sugar b eet r o o ts and le a v e s , and p os­ s i b l y pea sh o o ts c o n ta in ed IAA in s u f f i c i e n t q u a n t it ie s to be d e te c te d . DISCUSSION The r e s u lt s p resen ted in t h is stu d y i l l u s t r a t e some o f th e d i f f i c u l ­ t i e s in d e t e c tio n o f IAA in v e g e t a t iv e p la n t t i s s u e s . H ou sley, Booth and -6 2 - P h i l l i p s (20) rep o rted f a i l u r e to fin d IAA in corn s e e d lin g s , and our p r elim in a ry s t u d ie s confirm ed t h is fin d in g . R ein ert and Forstman (2?) o b ta in ed s im ila r r e s u l t s , and p o s tu la te d some s o r t o f b in ding o f IAA in a manner perhaps s im ila r to th a t in v e s t ig a te d by T eg e th o ff ( 3 6 ) in corn sc u te llu m . I t i s in t e r e s t in g th a t reco v ery o f 1 -3 jig o f IAA i s o n ly th e order o f 12 t o 14$; w h ile recovery o f 100 jag i s about 25 $ . The in c r e a se d r e c o v er y when th e aqueous co n cen tra te was made a lk a lin e su g g e sts some r e v e r s ib le b in d in g . o f IAA a t pH 4 .0 i s a f a c t o r . I t i s d o u b tfu l i f a c tu a l d e s tr u c tio n Brian (6) has found b in d in g o f 2 -m eth y l- 4-ch loroph en oxy a c e t i c a c id i s g rea ter a t low pH and th a t crude corn e x t r a c ts bind com p a ra tiv ely la r g e amounts. f r e e IAA e x i s t s in v iv o . One may q u e stio n whether any Though IAA has been d e te c te d in Avena c o l e o p t ile s (3 0 ,3 ? )» experim ents w ith r a d io a c t iv e IAA in d ic a te no r e d is t r ib u t io n under th e in flu e n c e o f g eo - or p h o to tr o p ic s tim u la tio n ( 7 > 9 ,1 4 ,2 8 ). From th e p r e se n t work, th e occu rren ce o f a p p r e c ia b le f r e e IAA in e th a n o lic e x tr a c ts o f corn sh o o ts must be co n sid ered d o u b tfu l, s in c e added IAA (1 -3 Mg) i s la r g e ly l o s t . One co u ld con clud e th a t f r e e IAA does occur in v i v o , but i s bound to some e th a n o l s o lu b le component upon grin d in g th e t i s s u e . A more a t t r a c t i v e p o s s i b i l i t y i s th a t a l l th e IAA occu rs in th e form o f a v e r y l a b i l e com plex. jig p er k ilogram . A lk a lin e eth er e x tr a c tio n le d to an e stim a te o f 1 3 .3 U sing t h i s v a lu e , one may estim a te th a t about 2 jag should have been d e te c te d ( a t l e a s t q u a lit a t iv e ly ) in th e experim ents u sin g a c id e th er e x t r a c tio n . T his was not th e c a s e , however. T h erefore, i t i s p rob ab le th a t endogenous IAA was lib e r a t e d from some s o r t o f complex in th e c a se o f a lk a lin e eth e r e x t r a c tio n . The p resen ce o f a complex could e x p la in th e experim ents on trop ism s dem onstrating a la c k o f r e d is t r ib u tio n o f r a d io a c t iv e IAA. -6 3 V lit o s and Meudt (4 0 ) have a ls o f a i l e d to f in d a p p recia b le amounts o f f r e e IAA in eth a n o l e x t r a c t s o f s e v e r a l p la n t s . The g r e a te r ea se w ith which IAA i s d e te c te d by wet eth er e x tr a c tio n may be due in p art to i t s a b i l i t y to d i s s o c i a t e th e bound IAA. In t h i s r e s p e c t, i t i s in t e r e s t in g to n o te th a t eth e r can p rev en t p olar auxin tra n sp o rt (39)* However, Wildman and Muir (43) have a ls o dem onstrated IAA p rodu ction from tryptophan during wet eth er e x t r a c tio n , and t h e ir p rod uction had a tem perature optimum o f 13°C. In th e p r e se n t work, IAA could be d e te c te d in corn sh o o ts or r o o ts by w et eth e r e x tr a c tio n fo r 3 hours a t 4°C. Furtherm ore, subsequent 70$ e th a n o l e x t r a c tio n o f th e r e s id u e r e s u lt e d in d e t e c tio n o f IAA. However, no IAA co u ld be d e te c te d in eth a n o l or subsequent eth e r e x tr a c ts when th e i n i t i a l e x tr a c tio n was w ith 80$ e th a n o l. There i s now no doubt th a t IAA can be d e riv e d from tryptophan in v it r o by p rep a ra tio n s from v a r io u s t i s s u e s ( 1 0 ,1 5 ,3 2 ,4 2 ,4 4 ) . I t must be m entioned th a t th e pro­ d u ctio n o f IAA from tryptophan in v it r o by a p a r t ic u la t e f r a c tio n from corn s e e d lin g s was a ls o ob served ( 1 8 ). When tryptophan-2-C s u b s tr a te , ca rb o x y l la b e le d IAA was o b ta in ed . 14 was used as The a c id eth er s o lu b le p rod u cts found were s im ila r to th o se rep orted by Dannenburg and Liverman (1 0 ). However, in c o n tr a s t to th e r e s u lt s o f t h e s e w orkers, l i t t l e la b e le d IAA cou ld be i s o l a t e d when e x c is e d corn sh o o ts were fe d tr y p to phan-2-C 14 In c o n tr a s t to th e growing sh o o t, corn k e r n e ls were found t o con­ t a in c o n s id e r a b le q u a n t it ie s o f f r e e IAA a s has been rep orted ( 3 ,8 ,1 6 ,3 2 , 3 3 ,4 0 ,4 4 ) . In a d d itio n , a w a t e r -in s o lu b le , e th a n o l-s o lu b le f r a c t io n , presum ably i d e n t i c a l to th a t o f Berger and Avery ( 3 , 4 ) , was p r e s e n t. T h is su b sta n ce m ight be a p r o te in or p o ly p e p tid e s in c e i t appears to be -6 4 p r e c ip it a t e d w ith ammonium s u l f a t e . I t should be n o ted , however, th a t S t e h s e l (3 2 ) found th e a l k a l i l a b i l e IAA complex in immature sweet corn seed was d ia ly z a b le . I t has been su g g ested t h a t tryp to p h a n -co n ta in in g p r o te in s in g en era l y i e l d IAA upon a lk a lin e h y d r o ly s is . Gordon (13) and Schocken (29) found th a t when p r o te in s were h yd rolyzed w ith a l k a l i , a su b sta n ce , presum ably IAA, c a u sin g s tim u la tio n in th e Avena curvatu re t e s t , cou ld be is o la t e d . The amount o f s tim u la tin g su b stan ce ob tain ed was c o r r e la te d w ith th e tryptophan c o n ten t o f th e p r o te in . On th e oth er hand. Wildman and Bonner (41) found a s p e c i f i c f r a c t io n o f spinach l e a f p r o te in co n ta in ed bound IAA. In th e p r e se n t exp erim en ts, n e ith e r tryptophan i t s e l f nor th e a lc o h o lin s o lu b le , w a te r -s o lu b le p r o te in o f corn g ra in formed IAA upon m ild a lk a lin e h y d r o ly s is ; but th e a lc o h o l- s o lu b le , w a te r -s o lu b le f r a c t io n d id . A number o f E h r lich r e a c t iv e sp o ts were d e te c te d in oth er p la n t t i s s u e s , but i t may not be concluded th a t a l l th e s e a re in d o le compounds. As has r e c e n t ly been p o in te d o u t, a number o f p h en ols g iv e p u rp le, red and pink c o lo r s (31)* I t was found th a t th e d e scrib e d eth er-w a ter p a r t it io n column some­ tim es f a i l e d as a good p u r i f i c a t io n s te p when th e a lk a lin e eth er e x tr a c tio n c o n s t it u t e d th e f i r s t p u r if ic a t io n s te p . This was a ls o noted during i s o l a t i o n o f IAA from U s tila g o Zeae tumors ( 3 8 ). In th e s e c a s e s , a l l th e IA A -2-C ^ appeared ju s t behind th e fr o n t in h ig h ly pigm ented f r a c t io n s . S in c e i t was found th a t f a i l u r e was n ot due to any change in th e pH o f th e aqueous p h a se, i t i s p rob ab le th a t p o la r e t h e r -s o lu b le m a te r ia ls p rev en ted th e proper p a r t it i o n . A p r io r p a r t it io n between n -hexan e/ a c e t o n i t r i l e d id not p rev en t t h is d i f f i c u l t y . In th e s e c a s e s , i t was u s u a lly n e c e ssa r y to chromatograph one or two tim es a s a 20 to 40 cm -6 5 s tr e a k , and f i n a l l y e le c tr o p h o r e se th e products as a 5 to 10 cm s tr e a k , a s d e sc r ib e d above. SUMMARY Methods found u s e f u l in th e i s o l a t i o n o f IAA from kilogram q u a n ti­ t i e s o f p la n t t i s s u e a re d e sc r ib e d . A b u ffered eth er-w a ter p a r t it io n column, e le ctr o p h o resis-ch ro m a to g ra p h y , and continu ous flow e le c t r o ­ p h o r e s is te c h n iq u e s were d evelop ed . The u se o f tr a c e amounts o f IAA14 2-C was e s p e c i a ll y v a lu a b le during th e i s o l a t i o n , and in e v a lu a tin g th e reco v e ry . Free IAA was d e te c te d in 80$ eth a n o l e x tr a c ts o f corn k e r n e ls and s h o o ts , as w e ll as v e g e t a tiv e sugar b e e t r o o ts and le a v e s . The d i f f i c u l t i e s o f d e t e c tio n o f IAA in corn sh o o ts are d isc u s se d and i t i s su g g e sted IAA may occur as a l a b i l e complex in eth an ol e x t r a c ts o f th is t is s u e . An 80$ e t h a n o l- s o lu b le , a q u e o u s-in so lu b le f r a c t io n from corn k e r n e ls was found to r e le a s e IAA upon a lk a lin e h y d r o ly s is . I t appears t h i s f r a c t io n may be p r o te in in n a tu re. We should l i k e to ta k e t h i s o p p o rtu n ity t o thank Dr. S. A. Gordon, who su p p lied th e IA A -2-C ^; and Dr. H. S e l l fo r u se o f th e vacuum spray dry apparatus. I t i s a p le a su r e t o acknowledge th e a s s is t a n c e o f Dr. H. Fukui in th e eth er e x tr a c tio n exp erim ents. -6 6 - TABLE I The R ecovery o f IA A -2-C ^ from E t io la te d Corn and Pea Shoots and th e D e te c tio n o f IAA in Corn Shoots jug IAA D e te c te d / Kg by S alkow ski jug IAA-2-C1^ $ Recovery Assay C orrected Added f o r Recovery o f IAA-2-C1^ T issu e Fresh Wt. Corn sh o o ts 1000 1 .8 9 Pea sh o o ts 950 0 .9 5 1 0 .7 Corn sh oots 1000 1 .8 9 1 4 .5 0 Corn sh o o ts 1000 2 .8 4 1 2 .4 0 Corn sh o o ts 1000 1 .8 9 1 2 .0 0 Corn sh o o ts 1000 2 .8 4 1 6 .1 0 844 2 .8 4 2 6 .0 0 Corn sh oots* 0? 0 Trace? 1 .6 5 2 8 .1 3000 Corn sh o o ts* 1 3 .3 * A lk a lin e e x tr a c tio n o f aqueous co n cen tra te w ith eth e r i n i t i a l l y . TABLE I I The Amount o f Free IAA I s o la t e d per Kilogram o f Corn G rain, and th e Amount o f IAA O btained by H y d ro ly sis o f th e 80$ E thanolS o lu b le , Water I n s o lu b le R esid ue fo r Two S u c c e s s iv e One Hour P erio d s w ith 1 N KOH a t 100°C Experiment-^ Free ju.g o f IAA/Kilo gram 2nd H yd rolysate 1 s t H yd rolysate 1 82 186 98 2 115 509 63 3 171 ------- 4 330 ------- — — — 52 1 6 ,8 0 0 ■^In experim ents 1 - 4 , M ichigan 350 grain was soaked fo r 4 hou rs. In experim ent 1 , i t was ground a f t e r soak in g; in 2 and 3 . i t was germin­ a te d 12 hours a t 25°C; and in 4 , germ inated 24 hours a t 25°C. ^Market s ta g e y e llo w sw eet corn k e r n e ls purchased l o c a l l y . -6 7 table in The Rf o f I s o la t e d and S y n th e tic IAA in 4 S o lv e n ts , and i t s M igration a f t e r E le c tr o p h o r e s is in 0 .1 M C itr a te B u ffer pH 5 .0 1 S o lv e n t Isop rop an ol; NH^OH; ^ 0 Rf (8 * 1 :1 ) 0 .3 7 P y r id in e ; NH^OH (4 s i ) 0 .6 2 n -B u tan ol; E thanol; HgO (4 :1 s i ) 0 .8 5 70$ E thanol O.7 8 2 .9 4 x (E le c tr o p h o r e s is ) " M igration cm ^/V olt-Sec. IQ "5 4.0 jug o f i s o l a t e d IAA TABLE IV The Area o f E h rlich R e a c tiv e Spots Follow ing^ Chromatography o f S y n th e tic and I s o la t e d IAA Area (Sq. Inch) Amount and Source o f IAA 10 jug S y n th e tic 1 .3 2 10 jig I so la te d ^ 1 .4 1 5 jug S y n th e tic + 5 jug I s o la t e d 2 1 .5 5 1 .6 3 10 jig S y n th e tic 10 jug I s o la t e d 1 s t H yd rolysate 2 1 .3 0 0 5 jug S y n th e tic + 5 jug 1 s t H yd rolysate1^ 1 .2 4 P lo t was 0 .1 in ch per 0 .1 in ch on f i l t e r paper, and 0 .1 in ch per 2$ tr a n sm issio n on v e r t i c a l a x i s . Beckman DU S pectrophotom eter, s t r i p d r iv e , 600 2 S alkow ski e stim a te s - 68- TABLE V Growth Promoting A c t iv i t y o f th e I s o la t e d "Free" IAA from Experiment 1 (T able I ) Compared t o That o f Known IAA in th e Avena S e c tio n T est Amount and Source o f IAA In c . in E lon s. (mm) No. Measured 1 ppm i s o l a t e d 1 ppm s y n th e tic 2-97 3 .2 0 24 21 0 .1 ppm i s o la t e d 0 .1 ppm s y n t h e t ic 3 .2 6 2 .9 1 25 26 0 .0 1 ppm i s o la t e d 0 .0 1 ppm s y n th e tic 1 .9 4 1 .7 7 26 25 C ontrol 0 .9 9 21 ‘'"Victory o a t s , i n i t i a l s e c t io n le n g th 6 .5 mm* f lo a t e d on 0 .0 1 M phos. ph a te b u ffe r pH 4 .5 w ith 2$ su c ro se a t 25°C fo r 10 hours. -6 9 table VI E h r lic h R e a c tiv e Su b stan ces D etected on Paper Chromatograms F ollow in g Column Chromatography o f th e A cid Ether F r a c tio n . Four 30 nil F r a c tio n s Were C o lle c te d From th e Column and Chromatographed S e p a r a te ly on Paper Fresh _____________ P la n t_______________ Wt. __________ Sub sta n ces Found Rf & Color w ith E h rlich *s Reagent* Sugar b e e t s , r o o ts* * 920 IAA Sugar b e e t s , le a v e s * * 1400 B lue 0 .3 2 6 ^ , IAA ( t r a c e ) ^ ’^, pink O.7 6 . Sugar b e e ts, flo w e r in g tops** 1100 Pink 0 .5 0 3 ’t Banana 1000 No E h rlich r e a c t iv e sp o ts (Aq. h y d r o ly sa te a f t e r Et 20 e x t .) ( 3 .8 Aig as e s t . by Salkow ski) L t. b lu e 0 . 2 7 . Cucumber, f r u i t 1500 No E h rlich r e a c t iv e s p o ts . P o ta to e s 1000 Blue changing to green 0 .3 3 . 950 Pink to p u rp le 0.32^*^ , pink fa d in g 0 .6 9 -0 .8 9 ^ * 3 . Pea s h o o ts , e t i o la t e d Green p e a s , market sta g e* * 1000 (Aq, h y d r o ly sa te a f t e r Et20 e x t .) Blue 0f 367^» pink 0 .4 3 ^ , pink 0.883 Blue 0 .5 1 2 , pink 0 .4 3 ^ , pink 0 . 883 *4'. * 1 ,2 ,3 and 4 a re c o n s e c u tiv e 30 ml column f r a c tio n s s t a r t in g w ith th e f r o n t , and Rf v a lu e s a re fo r isopropanol/am m onia/w ater ( 8 : 1 : 1 , v / v ) . ** A p r elim in a ry a lk a lin e eth er e x tr a c tio n was used in th e s e c a s e s . -7 0 - F igu re 1 The e f f e c t o f m o la r ity o f th e potassiu m phosphate b u ffe r (pH 6 .5 ) on th e m ig r a tio n o f IAA and corn sh oot pigm ents on a 10 gram C e lit e column 25 mm in d ia m eter. The boundary l i n e s in d ic a te q u a l i t a t i v e l y where e lu t io n o f IA A -0-0, and pigm ents - x - x b egin and end. F igu re 2 The e f f e c t o f pH on th e m ig ration o f IAA and corn shoot pigm ents on a 10 gram C e lit e column 25 mm in d iam eter. The boundary l i n e s in d ic a te q u a l i t a t i v e l y where e lu t io n o f IAA-0-0, and pigm ents - x - x b eg in and end. -7 1 - F ig . 1 1.0 - MOLAR PIGMENTS 0 .5 _ 0.1 15 ML ELUTED F ig . 2 6 .5 0 PIGMENTS IAA 6. 0 a. 5 .5 ■ MW I | ■ Pmi --— Til M - 10 15 ML ELUTED 20 25 30 -7 2 LITERATURE CITED 1. B e n tle y , Joyce A ., H ou sley, S . , and B r it t o n , G. Hormones and hormone p recu rso rs in le a v e s , r o o ts and s e e d s. Chem. and Mode o f A ctio n o f P la n t Growth S u b sta n ces. 4 0 -5 1 . (R. L. Wain and F. Wrightman, E d.) Academic P r e s s , N. Y. 1956. 2. B e n tle y , Joyce A. The n a tu r a lly -o c c u r r in g auxins and in h ib it o r s . Ann. Rev. P la n t P h y s io l. 9*47-80. 1958* 3* B erg er, J. and A very, G. S . I s o la t io n o f an auxin p recu rsor and an a u xin (I n d o le a c e t ic A cid) from m aize, Am. J. B o t. 31*199-203. 1944. 4. B erger, J . , and A very, G. S . 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Wain and F. Wrightman, E d.) Academic P r e s s , N. Y. 1956. 9. Ching, T. M ., and Fang, S . C. The r e d is t r ib u t io n o f r a d io a c t iv i t y in g e o t r o p ic a lly stim u la te d p la n ts p r e tr e a te d w ith r a d io a c tiv e in d o le ­ a c e t i c a c id . P h y s io l. P la n t. 1 1 :7 2 2 -7 2 3 . 1958. 10. Dannenburg, W. N . f and Liverman, J . L. C onversion o f tryp top h an -2Cl^ to in d o le a c e t ic a c id by waterm elon t i s s u e s l i c e s . P la n t P h y s io l. 3 2 :2 6 3 -2 6 9 . 1957* 11. Durrum, E. L. Continuous e le c t r o p h o r e s is and io n o p h o r e sis on f i l t e r p ap er. J. Am. Chem. S oc. 73*4875-4880. 1951® 12. F is c h e r , A ., and B ehrens, M. Versuche zur Trennung von In d o le d e r iv a ten aus w £ ssrig en P fla n zen ex tra k ten an der "auf s te ig e n d e n 11 C e llu lo s e s S u le . H oppe-Seylers Z. 291*242-244. 1953* 13. Gordon, S . A. Auxin p r o te in com plexes o f wheat g r a in . 33:160-169* 1946. Am. J. B ot, -7 3 - 14. Gordon, S , A ., and Eib* M. Auxin tr a n sp o r t in th e p h o to tro p ic r e sp o n se . P la n t P h y s io l. (Supp.) 31 XVI. 1956. 15. Gordon, S, A ., and Sanchez, N ieva F. The b io s y n t h e s is o f auxin in th e v e g e t a t iv e p in e a p p le , I I , The p recu rso rs o f in d o le a c e tic a c id . Arch, Biochem. 20?367-395- 19^9- 16. H aagen-Sm it, A. J . , D a n d lik er, W, B . , W ittw er, S. H, and Murneek, A. E. I s o l a t io n o f 3 - in d o le a c e t ic a c id from immature corn k e r n e ls . Am. J. B ot. 3 3 j1 1 8 -1 2 0 . 1946. 17*. H aagen-Sm it, A. J . , Leech, W. P. and Bergen, W. R. E stim a tio n , i s o l a t i o n and i d e n t i f i c a t i o n o f au xin s in p la n t m a te r ia l. Am. J. B ot. 2 9 :5 0 0 -5 0 6 . 1942. 18. H am ilton, R. H ., U npublished Data 19. H o lle y , R. W ., B o y le , F. P . , D u rfee, H. K. and H o lle y , A. D. A stu d y o f au xin s in cabbage u sin g cou n ter cu rren t d is t r ib u t io n . Arch. Biochem. B iop h ys. 32s1 9 2 -1 9 9 . 1951- 20. H ou sley, S . , Booth, A ., and P h i l l i p s , I . D. J. S tim u la tio n o f c o l e o p t i l e and r o o t growth by e x t r a c ts o f m aize. N ature. 1 7 8 s 2 5 5 -6 . 1956. 21. Kramer, M. and Went, F. W. The n ature o f auxin in tomato stem t i p s . P la n t P h y s io l. 2 4 s207-221. 1949. 22. Kdgl, F . , H aagen-Sm it, A. J. and E rxleb en , Hanni. S tu d ien fiber des vorkommen von Auxinen in m ensch lichen und in t ie r is c h e n Organismus. H oppe-Seylers Z. 2 2 0 ;1 3 7 -1 6 1 . 1934. 23. L arsen, P. On th e se p a r a tio n o f a c id ic and n o n -a c id ic au xin . P h y s io l. P la n t. 8:34 3 -3 57- 1955. 24. L in se r , H.V ersuche zur chrom atographisehen Trennung p f la n z lic h e r W u ch ssto ffe. P la n ta 3 9 0 7 7 -^ 0 1 . 1951- 25. McRae, D. H ., F o s te r , R. J. and Bonner, J, K in e tic s o f au xin in t e r ­ a c t io n . P la n t . P h y s io l. 28:343-355* 1953* 26. M artin, A. J. P. and Synge, R. L. M. A new form o f chromatogram em ploying two liq u id p h a se s. I . A th eo ry o f chromatography. Biochem. J . 3 5 :1 3 5 8 -1 3 6 8 . 1941. 27. R e in e r t, J. and Forstmann, E. U ntersucheingen fiber das Auxin der M a is -K o le o p tile . P la n ta 52?623-628. 1959- 28. R e is e n e r , H. J. and Simon, H. W eiter G eotropism us-V ersuche m it r a d io a k tiv e r £ - I n d o ly le s s ig s & u r e (IE S -2-C l^ ) Z. B otanik 48? 66- 70. I960. 29- Schocken, V. G enesis o f auxin during d ecom p osition o f p r o t e in s . Arch. Biochem. 23? 198-204'. 1949- -7 4 30, Shibaoka, H. and Yamaki, T. A s e n s it iz e d Avena cu rvatu re t e s t and i d e n t i f i c a t i o n o f th e d i f f u s i b l e auxin in Avena c o l e o p t il e . The B ot. Mag. Tokyo 7 2 s l5 2 -1 5 8 . 1959. 31® S t e e lin k , C. C olor r e a c tio n o f c e r ta in p h en ols w ith E h r lic h 's r e a g e n t. N ature 184s 720. 1959. 32. S t e h s e l, M. L. S tu d ie s on th e nature o f th e auxin complex in th e corn k e r n e l. Ph.D. T h esis Univ. o f C a lif . (B e r k e le y ). 1950, 33® Stow e, B. B. and Thimann, K.V. The paper chromatography o f in d o le compounds and some in d o le c o n ta in in g au xin s o f p la n t t i s s u e . Arch. Biochem. B io p h y sic s 51$499-515® 1954. 34. S t u t z , R. E . , A tk in so n , D. E. and Gordon, S. A. L a b o ra to ries Report No. 4710. 1951. 35® Tang, Y. W. and Bonner, J. The enzym atic in a c t iv a t io n o f IAA. I . Some c h a r a c t e r is t ic s o f th e enzyme co n ta in ed in pea s e e d lin g s . Arch. Biochem. 1 3 s1 1 -2 5 . 1947. 36. T e g e th o ff, B. Zur Frage der W u ch ssto ffin a k tiv ieru n g im M a iss c u te llum, P la n ta 3 8 s6 4 8 -6 8 2 . 1951® 37® T e r p stra , W. E x tr a c tio n and i d e n t i f i c a t i o n o f p la n t growth sub­ sta n c e s o Ph.D. T h e sis B o ta n ic a l L a b o ra to ries, U trech t. 1953® 38. T urian, G. and H am ilton, R® H. I s o la t io n o f 3 - in d o ly la c e t ic a cid from U s tila g o zeae tum ors. Biochem. e t B io p h y sics Acta ( in p r e s s ) . 39® Van Der W eji, H. G. Der mechanismus des W u ch ssto fftra n sp o rtes I I . Rec. Trav. B ot. N e e r l. 31$810=857® 1934. 40. V l i t o s , A. J. and Meudt, W„ The r o le o f auxin in p la n t flo w e r in g . I . A q u a n tit a tiv e method based on paper chromatography fo r th e d eterm in a tio n o f in d o le compounds and IAA in p la n t t i s s u e . C on trib . Boyce Thompson I n s t . 1 7 s1 9 7 -2 0 2 . 1953® 41. Wildman, S. G. and Bonner, J. The p r o te in s o f green le a v e s . I . I s o l a t io n enzym atic p r o p e r tie s and auxin co n ten t o f spin ach c y to ­ p la sm ic p r o t e in s . Arch. Biochem. 1 4 s381-413. 1947. 42 . Wildman, S . G ., F e r r i, M. G. and Bonner, J. The enzym atic c o n v ersio n o f tryptophan to auxin by spin ach le a v e s . Arch. Biochem. 13$131-134. 1947. 43. Wildman, S . G, and Muir, R. M. O bservation on th e mechanism o f au xin form ation in p la n t t i s s u e s . P la n t P h y s io l. 2 4 s8 4-92. 1949. 44. Yamaki, T. and Nakamura, K. Formation o f in d o le a c e t ic a c id in maize embryo. S c i . Papers C o ll. Gen. E d ucation, Univ. Tokyo 2;81= 9 8 . 1952. Argon N a tio n a l -7 5 3. In V ivo L ab elin g Experim ents on th e B io s y n th e s is o f IAA Though th e i s o l a t i o n work in d ic a te d th a t corn sh o o ts co n ta in ed a m inute amount o f IAA, i t was co n sid ered th a t IAA m ight be a f le e t i n g in te r m e d ia te and never accum ulates in r a p id ly growing t i s s u e . Thus th e o n ly p o s s i b i l i t y o f dem onstrating i t s p resen ce would be to trap th e IAA in some manner. I t was thought th a t corn s e e d lin g s should be a b le to f i x CO2 in th e dark v ia th e phospheonolpyruvate ca rb o x y la se (1 1 ) or some o th er c a r b o x y la tin g r e a c tio n . In e it h e r c a s e , th e carbon i s f ix e d in to o rgan ic a c id s which are v ery a c t iv e m e ta b o lite s . I t was 14 hoped t h a t by such fe e d in g o f C C>2 th a t th e s id e chain o f IAA would become la b e le d . In an attem pt to get accum ulation o f th e la b e le d IAA, u n la b eled IAA was used a s a tr a p . In th e f i r s t experim ent, 200 grams o f corn sh o o ts were fe d 1 m ill i c u r i e o f C-^ 0 2 and 100 jum o f IAA during 8-hour p e r io d in th e dark. The sh oots were washed and th e IAA i s o l a t e d . There was no r a d i o a c t iv i t y in th e is o la t e d IAA, and th u s CO2 was e it h e r a poor p r e c u r so r , or no IAA was sy n th e s iz e d during th e experim ent. In c o n sid e r in g th e l a t t e r a l t e r n a t iv e , i t was thought th a t th e added IAA m ight have p rev en ted i t s s y n t h e s is in v iv o . A second experim ent u sin g 500 grams o f corn sh o o ts and fe e d in g 100 juM o f in d o le a s w e ll as 1 me o f C-^02 was run f o r 4 hours in th e dark. Thus in t h is experim ent a p re­ cu rsor o f IAA was fe d in an attem pt to s tim u la te i t s s y n t h e s is . added a s c a r r ie r p r io r to i s o l a t i o n . IAA was The is o la t e d IAA had 8 counts per m inute above background a f t e r chromatography once. I t was then run on th e con tin u ou s flo w eL ectrop h oresis u r it , but was l o s t due to poor pH ad justm en t b e fo r e running. Tryptophan was i s o l a t e d by a d so r p tio n on an IR 120 exchange column in mixed Na and H form a t pH 3*1» and e lu t io n w ith 0 .1 M NH^OH. For paper -7 6 chrom atography, th e volume was reduced in vacu o, and th e sample s p o tte d on Whatman 1 paper u sin g isopropanol/am m onia/w ater ( 8 :1 : 1 ) s o lv e n t a sce n d in g . The tryp top h an area was e lu te d and rechrom atographed u sin g 77$ eth a n o l a s th e s o lv e n t . The tryptophan was assa y ed by th e q u a n ti­ t a t i v e n in h yd rin r e a c tio n o f Mooreand S t e in (109)® o f th e tryp top h an so i s o l a t e d was 157 cpm/juM. The s p e c if i c a c t i v i t y The t o t a l f r e e tr y p to ­ phan i s o l a t e d was 625 micrograms as estim a ted by n in h yd rin a ssa y (3®06 m icro m o les). S in c e th e f r e e tryptophan has such low a c t i v i t y ( l e s s than 1 cpm/jag) th e a c t i v i t y in IAA would n ot have been d e te c te d , assum­ in g a d ir e c t c o n v er sio n . Only a tr a c e o f IAA would be ex p ected to be p r e se n t and i t s s p e c i f i c a c t i v i t y would be ex p ected to be l e s s than tryptophan due to l o s s o f th e term in a l carbon. Thus under th e s e con­ d i t i o n s , 0*^0g i s n ot a s u it a b le p recu rsor fo r e it h e r tryptophan or IAA. A f i n a l C-^Og la b e lin g experim ent was t r ie d in th e l i g h t . E igh t hundred and tw en ty grams o f 5 -d a y -o ld corn sh o o ts were fe d 1 me 3 —4 10”^ M in d o le , and 10 M IAA fo r 6 hours under d if f u s e window l i g h t . A fte r f lu s h in g th e o u t o f th e m od ified d e s ic c a to r in to a l k a l i , th e corn sh o o ts were washed and ground in s u f f i c i e n t 95$ eth a n o l to make th e f i n a l c o n c e n tr a tio n about 80$. The IAA and tryptophan were i s o l a t e d and t h e ir r a d i o a c t iv i t y compared by cou nting th e d rie d 50 $ eth a n o l e lu a n ts from paper chromatograms. The u su a l i s o l a t i o n procedure fo r IAA was em ployed, but th e i s o la t e d IAA was rechrom atographed tw ic e to co n sta n t s p e c i f i c a c t i v i t y . Tryptophan, i s o la t e d as d e sc r ib e d above, was a ls o rechrom atographed tw ic e in an attem pt to reach co n sta n t s p e c if ic a c t iv it y . Under t h e s e c o n d itio n s (T able I ) , th e a c t i v i t y o f IAA i s 9®54 cmp compared t o a f i n a l a c t i v i t y o f th e tryptophan o f 0 .3 8 2 cpm/jug. -7 7 Assuming th e IAA and tryp top han were o f about th e same s p e c if i c a c t i v i t y t h i s would mean t h a t 2 4 ,6 jag o f IAA must have been p r e s e n t. S in c e t h i s was not p o s s ib le , th e IAA may have a much h igh er s p e c if i c a c t i v i t y than th e tryp top h an , in which ca se i t cou ld n ot have come from th e tryp top h an . A try p to p h a n -2 - fe e d in g experim ent was n ext conducted in order to d eterm ine i f s ig n i f i c a n t amounts o f IAA cou ld be ob tain ed from la b e le d tryp top h an . A t o t a l o f 3 • 730*000 cpm o f tryp top h an -2 - ( s p e c i f i c a c t i v i t y = 0.42juc/juM) was fe d fo r 6 hours t o 500 g o f corn s h o o ts . The t o t a l a c t i v i t y in th e IAA f r a c t io n was 49*5 cpm. S in ce th e tryptophan had a s p e c i f i c a c t i v i t y o f 17^5 cmp/jag, o n ly 0 .0 2 3 J^g o f IAA was formed from th e la b e le d tryptophan under t h e s e c o n d itio n s . However, th e sh o o ts c o n ta in 1 or 2 mg o f f r e e tryp top h an , and s in c e perhaps o n ly 10$ o f th e tryp toph an-2-C -^ en tered th e s h o o ts , th e in te r n a l tryptophan p o o l m ight have had a s p e c if i c a c t i v i t y o f about 0 .1 or 0 ,0 5 th a t o f th e tryptophan fe d . Thus th e IAA c o n c e n tr a tio n may have a c t u a lly been 0 .2 3 t o 0 . 4 6 ^ug. I t i s apparent th a t i n s i g n if ic a n t amounts o f IAA a re formed from e it h e r tryptophan or CO2 even though an enzym atic c o n v ertin g system fo r tryptophan may be dem onstrated in v i t r o . In view o f th e extrem ely sm all amounts o f IAA in th e corn s h o o ts , and th e r e l a t i v e l y g re a t l o s s e s during i s o l a t i o n , i t i s not s u r p r is in g th a t th e s e in v iv o la b e lin g experim ents were not e n t ir e ly s u c c e s s f u l. -7 8 TABLE I R a d io a c t iv it y o f I s o la t e d Tryptophan and IAA From 820 Grams o f Corn Shoots Fed 10-3 M I n d o le , 1 me and 10"^ M IAA fo r 6 Hours in th e L igh t Counts/ Min, » C ounts/ Min. Zju£ T otal jug I s o la t e d 1 3 .4 0 .0 6 1 220 Rechromatography 77$ e th a n o l 6 ,0 0o043 140 Rechromatography p y r id in e , ammonia ( 4 / l ) 2 .6 0 .043 60 IAA Tryptophan 6 , 0 1 3 .8 1 .4 7 4100 Rechromatography a c e to n e , ch loroform , ammonia, w ater ( 1 50 / 25 / 2 0 / 1 ) 1 ,7 8 5 .9 0 .8 5 3 2091 2 1 3 .6 0.382 558 Rechromatography p y r id in e , ammonia ( 4 /1 ) -7 9 - B. S y n th e tic P la n t Growth Substances 1. The E f f e c t o f N -l-N ap h th ylphthalm ic A cid on th e Growth and Geo­ t r o p ic R esponse o f S e e d lin g s . K o e p fli, Thimann and Went (34), u sin g th e pea t e s t , form ulated th e s p e c i f i c s tr u c t u r a l requirem ents o f a r in g w ith a double bond, a s id e ch ain w ith a carb oxyl group, a t l e a s t one carbon atom removed from th e r in g , and th e carb oxyl group having a p a r tic u la r space r e la t io n to th e r in g . Many ex c e p tio n s o f a c t iv e compounds not m eeting th e s e req u ir e ­ ments are known. V e ld str a ( 1 6 7 ) , in in v e s t ig a t in g p o la ro ig ra p h ic r e ­ d u c tio n o f th e r in g double bond o f au xin found a su p p ression o f th e oxygen maximum. T his in d ic a te d some s o r t o f s e l e c t i v e su r fa c e adsorp­ t io n to th e mercury drop. High su r fa c e a c t i v i t y in t h i s t e s t c o r r e la te d f a i r l y w e ll w ith th e b e e t -r o o t s e c tio n p e r m e a b ility fo r chlorophenoxy a c e t i c a c id d e r iv a t iv e s . B o o ij and V e ld s tr a , as c i t e d by V eld stra (1 6 8 ), l a t e r in v e s t ig a t e d in t e r f a c e accum ulation between o l e i c a c id w ater. In a g en era l way, th e h yd rop h ylic l i p o p h il i c balan ce i s im portant fo r a c tiv ity . T his i s proposed to be due to th e a d so rp tio n o f th e l i p o - p h y lic r in g t o th e l i p o i d a l c e l l u l a r membrane. V eld stra (168) has s a id " su rfa ce a c t i v i t y comes to stand more and more fo r th e s p e c if i c ad so rp tio n a f f i n i t y fo r th e s i t e s in v o lv ed in g ro w th .11 Skoog, Sch n eid er and Malan (138) developed th e view o f s p e c if ic antagonism . They were a b le to dem onstrate Y -p h e n y lb u ty ric a c id (though a weak au x in ) in h i b it s a c t i v i t y o f IAA in th e Avena cu rvatu re t e s t , and t h i s in h ib it io n cou ld be reduced by in c r e a s in g IAA c o n c e n tr a tio n . S im ila r antagonism s cou ld be e s ta b lis h e d fo r tran scin n am ic a c id (1 6 6 ), p h en o x y iso b u ty ric a c id s ( 2 9 ) , and 1-n aph th yl s u lf id e p ro p io n ic a c id (1 ) -8 0 a s w e ll as o th e r s . McRae, Bonner and F o ster (4 2 ,1 0 5 ) in trod uced th e u se o f c l a s s i c a l enzyme k i n e t ic s fo r d e s c r ib in g c o m p e titiv e in h ib it io n in th e Avena s e c t io n t e s t . F o s te r , McRae and Bonner (42) co n sid ered growth in h ib it io n a t h igh c o n c e n tr a tio n s o f IAA to be due to a r e q u ir e ­ ment fo r two p o in t attach m en t. The exp erim ental data do f i t th e c a l­ c u la te d k in e t ic e x p r e ssio n fo r t h i s c a se f a i r l y w e l l. The most im­ p o r ta n t u t i l i t y o f t h i s method, however, was an a n a ly s is o f th e n ature o f in h ib it io n as w e ll as e s tim a tin g maximum v e lo c it y and Ks o f a g iv en growth su b sta n c e . The b a sic assum ption in enzyme k in e t ic s i s th e form ation o f a d is s o c ia b le enzyme su b s tr a te complex which can break up ir r e v e r s i b l y in to product p lu s enzyme. The t o t a l amount o f enzyme ( f r e e and bound) b ein g c o n sta n t during th e experim ent, and th e amount o f sub­ s t r a t e b ein g in e x c e ss ( i . e . amount bound i n s i g n if ic a n t and c o n s ta n t, or stea d y s t a t e a ssu m p tio n ), i t can be v is u a liz e d th a t under th e s e con­ d it io n s th e r a te o f r e a c tio n w i l l be enzyme lim it in g and lin e a r w ith tim e. S e v e r a l problems a r i s e in c o n sid e r in g a p p lic a tio n to th e in viv o system . Endogenous s u b s tr a te would le a d t o n o n -lin e a r r e c ip r o c a l p lo t s ( 1 2 5 ) , but in g en era l t h e s e a re n ot found ex p erim en ta lly fo r s e v e r a l a c t iv e growth su b sta n c e s. i s a ls o p o s s i b l e . In th e l i v i n g system , enzyme or s i t e s y n th e s is I t has been found th a t alw ays o b ta in ed (1 2 ,6 8 ) and t h i s was a ls o a lin e a r tim e cou rse i s not th e c a se in th e p r e se n t stu d y. H ou sley, B e n tle y and B ic k le (68) rep o rt th a t a p p recia b le amounts o f au xin e n te r through th e c u t ends o f f lo a t in g Avena s e c t io n s , and thus tr a n s lo c a t io n i s a f a c t o r . In a d d itio n , th ey rep o rt p e r m e a b ility can a ls o be a f a c to r on th e b a s is o f in d ir e c t I t has been p o in ted ou t by B o t t e lie r te sts. (22) th a t th e r e c ip r o c a l form o f th e enzyme k in e t ic eq u a tion i s fo rm a lly th e same as Langmuir's ad- -8 1 s o r p tio n iso th erm . He s u g g e sts th e growth o f c o l e o p t il e s e c tio n s i s b e t t e r f i t by lo g G = A + b lo g C, where G = growth and C = concen­ tr a tio n . Other growth t e s t s were b e t te r f i t by oth er em p irica l e q u a tio n s , and no one eq u a tion was s a t is f a c t o r y fo r a l l c a s e s . How­ e v e r , i t would appear th a t th e enzyme k in e t ic concept s t i l l g iv e s th e most in t e r e s t in g p h y s io lo g ic a l e v a lu a tio n o f th e data in c a se s where i t i s v a lid . In th e p r e se n t stu d y on t e t r a z o le a n a lo g u es, th e p resen ce o f two s i t e s o f a c tio n appears in d ic a te d due to th e f a c t th e 2,4-D an alogu e a cte d c o m p e titiv e ly toward o n ly 2,4-D induced growth. In a d d itio n , th e cu rv a tu re in th e r e c ip r o c a l p lo t o f t h i s data appears s im ila r to th e c a s e fo r two enzymes d is c u s s e d by R einer (1 2 5 ). R ein er (125) p o in ts o u t, however, th a t com bination o f th e in h ib i­ t o r w ith th e s u b s tr a te or co-enzym e so a s to low er i t s co n c e n tr a tio n or r e s u lt in an in h ib it o r y com plex, a ls o r e s u lt s in cu rvatu re in th e r e c ip r o c a l p l o t . 2. B io lo g ic a l A c t i v i t y o f T e tr a z o le Analogues o f IAA and 2,4-D I t has now been e s ta b lis h e d th a t a number o f s y n th e tic growth su b sta n ces can s e l e c t i v e l y in h ib it both phototropism and geotropism in s e e d lin g s . Such e f f e c t s were f i r s t observed by Mentzer and N etien (1 0 6 ), but i t appeared n e c e ssa r y in our work to e s t a b lis h i t s r e la t io n to in ­ h i b i t i o n o f growth. The im portant work o f Jones, M etca lfe and Sexton (7 5 ) in d ic a te d a number o f compounds have t h e s e e f f e c t s . Henderson and P e te r so n (64) have found 100-1000 ppm 2,4-D to in h ib it th e photo­ t r o p ic and g e o tr o p ic resp o n se o f o a t c o l e o p t i l e s . They assume no in ­ h i b i t i o n o f s t r a ig h t growth, but p r e se n t no evid en ce on t h is p o in t. Vander Beek (164) f a i l e d to f in d an e f f e c t w ith 2 ,4 -D in h is o a t sh oot -8 2 t e s t a t low er c o n c e n tr a tio n s . He found 2 ,3 -6 - tr ic h lo r o - , 2 , 3 ,5 - t r i o d o - , and 2 , 6 -d ic h lo r o b e n z o ic a c id a c t iv e in in h ib it io n o f both p h o to tro p ic and g e o tr o p ic r e sp o n se , whereas a number o f o th er an alogs were in a c t iv e in both t e s t s . The ev id en ce appears stro n g th a t c e r t a in s y n th e tic com­ pounds a r e e s p e c i a ll y e f f e c t i v e in s e l e c t i v e in h ib it io n o f p h o to tro p ic or g e o tr o p ic r e s p o n se s . The p r e se n t work has been d isc u s se d by Morgan and Sflding (110) who r ep o rt NPA (N -l-n a p h th y lp h th alam ic a c id ) i s a c t iv e under t h e ir con­ d it io n s in th e Avena s e c t io n t e s t . In in t e r e a c t io n w ith low con cen tra­ t io n s o f IAA, i t fu r th e r stim u la te d growth. However, in th e cu rvatu re t e s t or c y lin d e r t e s t s , th e NPA in h ib it e d th e IAA growth resp o n se. It was su g g e ste d NPA, though a weak a u x in , in h ib it s p o la r tr a n sp o r t o f IAA. Thus in c y lin d e r growth t e s t s , s tim u la tio n induced by IAA (or NPA a lo n e ) was lim it e d t o f i r s t 1 or 2 mm, w ith th e more b a sa l zones showing in ­ h ib itio n . These au th ors su g g e st a l l ch em icals s e l e c t i v e l y in h ib it in g tro p ism s may d is tu r b p o la r tr a n sp o r t o f IAA. t r ib u t io n o f IAA-C 14 The la c k o f l a t e r a l r e d is - during g e o tro p ic or p h o to tr o p ic cu rv a tu re, however, h as a lr e a d y been d is c u s s e d . Morgan ( p r iv a te com munication) has in d ic a te d t h i s makes a l a t e r a l r e d is t r ib u t io n th eo ry u n a tt r a c t iv e . Anker ( 6 ,? ) has p r ese n te d evid en ce to show th a t geotrop ism has an optimum e q u iv a le n t to th e maximum a c c e le r a t io n o f growth and th a t con­ c e n tr a tio n s in d u cin g maximum growth ca u ses in h ib it io n o f geotropism . In h is t e s t s , d e c a p ita te d c o l e o p t il e s were p la ced in aqueous s o lu t io n s o f t h e growth su b sta n c e s. Anker ( 6 ,7 ,8 ) in a s e r ie s o f s t u d ie s , showed th a t IAN, IAA and NAA were a l l a c t iv e ( in d escen d in g order o f a c t i v i t y ) , in in d u cin g g e o tr o p ic cu rv a tu re in d e c a p ita te d c o l e o p t i l e s . Anker (6) assum es th a t in h ib it io n o f geotropism by high c o n c e n tr a tio n s i s due to -8 3 perm eation o f th e su b sta n ce through th e c u t i c l e r a th e r than th e t i p , r e s u lt in g in " flo o d in g ." DeWit (35) t u sin g A nker's tech n iq u e, found th a t IAA had to be p r esen t during g eo tr o p ic s tim u la tio n . "Flooding" occu rred when 1 mg per 1 ml was p resen ted to n o n -d eca p ita ted c o le o p ­ tile s . C urvatures co n tin u ed , when IAA was removed, fo r about 30 m inutes; in d ic a tin g d e p le tio n o f th e IAA. B o t t e lie r and R oosheral (23) in a sh o r t p ap er, used A nker's tech n iq u e to t e s t our assum ption th a t in h ib it io n o f growth and th e r e fo r e geotrop ism , has to be p r o p o r tio n a l. They used d ie t h y l eth er and found th e p ercen t o f in h ib it io n o f geo­ trop ism was more s e n s i t i v e than p ercen t in h ib it io n o f growth. They t h e r e fo r e s u g g e st an in h ib it io n o f l a t e r a l tr a n s p o r t, which was a ls o one o f our s u g g e s tio n s . Though th e Dutch workers uphold th e c l a s s i c a l v ie w p o in t o f l a t e r a l p o l a r i t y o f auxin t r a n s lo c a t io n , o th er ex p la n a tio n s are p o s s i b l e , and t h e s e have been m entioned a lr e a d y . SELECTIVE INHIBITION OF THE GEOTROPIC RESPONSE BY n-l-NAPHTHYLPHTHALAMIC ACID By Te-May Tsou Ching, Robert H. Hamilton and Robert S. Bandurski P h y s io lo g ia Plantarum , V o l. 9» 5^6-558, 1958 PH Y SIO LO G IA PLANTARUM , VOL. 9, 54 6 -5 5 8 , 1956 T E - M A Y T S O U C H I N G , R O B E R T H. H A M I L T O N and R O B E R T S. B A N D U R S K I Selective Inhibition of the Geotropic Response by /i-l-Naphthylphthalam ic Acid R eprinted from PH YSIO LO G IA PLANTARUM O fficial p u b lication o f the S can d inavian S ociety for P lan t P h y sio lo g y E d itorial o ffice: B o ta n ica l L ab oratory, L und, Sverige P u b lish ers: E. M unksgaard, N orregade 6, K obenhavn, D anm ark — C opyright reserved — P H Y S I O L O G IA P L A N T A R U M , V O L . 9. 1 9 5 6 S e le ctiv e I n h ib itio n o f th e G eo tro p ic R e sp o n se by n -l-N a p h th y lp h th a la m ic A c id 1 By T e -M a y T so u Ch in g , R o b e r t H. H a m il t o n 2 a n d R o b e r t S. B a n d u r s k i D e p a i'tm e n t o f B o t a n y a n d P la n t P a t h o lo g y M ic h ig a n S ta te U n iv e r s ity , E a s t L a n s in g , M ic h ig a n , U .S .A . (R eceived Ju n e 1, 1956) I n tr o d u c tio n A ccord ing to th e th eory o f W en t a n d G h olod n y (1937), th e geotropic resp on se o f p lant seed lin g s is d ue to d iffer en tia l g ro w th fo llo w in g an internal red istrib u tion o f au xin . S in ce g eotrop ic cu rv a tu re resu lts sim p ly from unequal grow th , a direct correlation b etw een g row th rate an d rate o f geotrop ic curva­ ture is to be exp ected an d an in h ib itio n o f g ro w th sh o u ld resu lt in a propor­ tion al in h ib itio n o f curvature. In p rev io u s stu d ies fro m th is laboratory (G rigsby e t al, 1954) it w a s fo u n d th a t trea tm en t o f p ea seed lin g s with n -l-n a p h th y lp h th a la m ic acid (NP) resu lted in a 70 p ercen t reduction of straigh t grow th an d a co m p lete lo ss o f g eo tr o p ic se n sitiv ity . N etien and C onillot (1951) h ad earlier m a d e a sim ilar, a lth o u g h q u a lita tiv e, observation. T h is d isp rop ortion ality o f grow th a n d g eo tro p ic resp o n se, if significant, w o u ld seem to require a m o d ific a tio n o f th e cla ssic a l th e o ry o f the tropic m ech a n ism . A stu d y w as th erefore in itia te d to ex a m in e th e se observations q u an titatively an d to exta n d them to several a d d itio n a l p lan t sp ecies. H o ffm a n n an d S m ith (1949) h ave ob serv ed g ro w th reg u la tin g effects of d erivatives of p h th a la m ic acid. T h ey stated th a t at 0.1 p pm , N P induced 1 R ep ort o f w ork su p p orted in p art b y th e M ich igan A gricu ltu ral E x p erim en t Station, and in part by the N a tio n a l S cien ce F o u n d a tio n . J o u rn a l a rticle 1888 from the Michigan A gricu ltu ral E xp erim en t S tation . - A gent o f F ield C rops R esearch B ran ch , A gricu ltu ral R esearch S erv ice, U nited States D ep artm en t o f A gricu ltu re. P h y s i o l . P l a n t . , 9, 1950 [5 4 6 ] SELECTIVE IN H IBITIO N OF THE GEOTROPIC RESPONSE BY NP 547 leaf-rolling in tom ato plants; at 0.31 ppm , ep in asty w as observed; and at 20 ppm , stem sw ellin g. M entzer, M olho, and P ach eco (1950) tested tw entyfive related com p ou n d s on lentil roots, and con clud ed that a close spatial relationship of the peptide b ond and a carboxyl group w as required for inhibition o f geotropism . M entzer and N etien (1950) studied the effect of NP on the geotropic resp onse of pea, lentil, crucifer, tom ato, sunflow er, and cucum ber seed lin gs, and fou n d that 10-3 to 10"6 M con centration s caused an apparent n egative geotropism . Treated tap roots w ere som ew h at thicker and shorter than the control. In a study of the effect of N P on germ ination and early stages o f grow th, N etien and Conillot (1951) claim ed that the chem ical retarded th e germ in ation of som e species and in hibited straight growth in m ost sp ecies tested. M aterials and m e th o d s C o m m e r c ia l g r a d e N P w a s r e c r y s ta lliz e d fr o m a c e t o n e - p e tr o le u m e th e r . F o r u se, the r e c r y s ta lliz e d m a te r ia l (m .p . 1 9 1 °) w a s c o n v e r te d to th e p o ta s s iu m sa lt b y the a d d itio n o f a s t o ic h io m e tr ic a m o u n t o f p o ta s s iu m b ic a r b o n a te in a q u e o u s -a c e to n e so lu tio n . T h e s a lt w a s r e c o v e r e d b y e v a p o r a tio n o f th e s o lv e n t in v a c u o at 3 7 ° C w ith e b u llitio n o f d ry n itr o g e n g a s. S p e c t o p h o t o m e tr ic a n a ly s is y ie ld e d a m o la r e x tin c tio n c o e f f ic ie n t o f E 284 — 1 .5 9 5 X 10 4. T h e s ta n d a r d A v e n a c o le o p t ile s e c tio n test (M cR ae, F o s t e r a n d B o n n e r , 1953) w a s u se d to d e te r m in e th e in te r a c tio n o f in d o le a c e t ic a c id (IAA) a n d N P . T h e sta n d a rd e r r o r o f e a c h te st w a s le s s th a n 10 p e r c e n t, a n d th e re w a s n o s ig n ific a n t d iffe r e n c e b e t w e e n r e p lic a te s . F o r str a ig h t g r o w th a n d g e o t r o p ic c u r v a tu r e te sts, in ta c t s e e d lin g s w e r e u sed . V icto ry o a ts (A v e n a s a t i v a v a r. S ie g e s h a fe r ) w e r e o b ta in e d th r o u g h th e c o u r te s y o f Dr. J. B o n n e r . H y b r id c o r n (Z e a m a y s , W 2 3 X O h 5 1 a , a sin g le c r o s s h y b r id ) w a s k in d ly s u p p lie d b y D r. E . R o s s m a n , D e p a r tm e n t o f F a r m C rop s, M ic h ig a n S ta te U n iv e r s ity . G a rd en p ea (P i s u m s a t i v u m v a r. A la sk a ) w a s o b ta in e d fr o m th e F e r r y M orse S eed C o m p a n y . S eed s w e r e g e r m in a te d in th e d a rk at 2 5 ° C a n d a ll s u b s e q u e n t o p e r a tio n s w ere co n d u c te d in s u b d u e d red lig h t. S e e d lin g s w ith s tr a ig h t ta p r o o ts a n d s h o o ts o f a d esired le n g th w e r e s e le c te d fo r t h e e x p e r im e n ts . A v e n a c o le o p t ile s a n d r o o ts o f co rn a n d p ea w e r e h a r v e s te d at 2 .0 ± 0 . 5 cm , w h ile s h o o t s o f c o r n a n d p ea w e r e u sed at 1 .2 5 ± 0 . 2 5 cm . S e e d lin g s w e r e p r e tr e a te d w ith N P or o th e r c o m p o u n d s b y im m e r s io n o f th e s e e d lin g s in a s o lu t io n o f th e c o m p o u n d d is s o lv e d in 0 .0 0 5 to 0.01 m o la r p h o s p h a t e b u ff e r at p H 4.5 . C o n tr o l p la n ts w e r e sim ila r ly trea ted in th e b u ffe r . P r e -tr e a tm e n t tim e f o r o a t c o le o p t ile s w a s 30 m in u te s a n d 60 m in u te s fo r b oth r o o ts a n d s h o o t s o f c o r n a n d p ea . A lo n g e r p r e -tr e a tm e n t tim e w a s u se d fo r co rn a n d p ea s e e d lin g s s in c e th e r e s p o n s e s u b s e q u e n t to 30 m in u te tr e a tm e n t w a s n o t u n ifo r m . A fte r p r e -tr e a tm e n t th e s e e d lin g s w e r e d iv id e d in to tw o g r o u p s; o n e fo r th e g e o t r o p ic te st, a n d th e o th e r fo r th e s tr a ig h t g r o w th test. F o r th e g e o tr o p ic test, p la n ts w e r e h e ld in a h o r iz o n t a l p o s it io n b y m e a n s o f an A v en a h o ld e r o r b y m e a n s o f p r e s s u r e s e n s itiv e ta p e w it h a d h e s iv e o n b o th sid e s . T h e ta p e w a s m o u n te d P h y s i o l. P la nt ., 0, 1956 548 T E -M A Y TSO U CHING, R O BE R T H. H AM ILTO N AND RO BE R T S. BAN DURSK I o n a m o is t b lo tt e r p a p e r a n d t h e s e e d s c o v e r e d w it h m o is t t is s u e p a p e r . F o r the s t r a ig h t g r o w t h t e s t , p la n ts w e r e h e ld in a v e r t ic a l p o s it io n b y t h e s a m e m ea n s. B o t h s e ts w e r e in c u b a te d a t 2 5 ° C a n d 9 0 p e r c e n t r e la t iv e h u m id it y . G e o tr o p ic c u r v a t u r e w a s m e a s u r e d f r o m s h a d o w g r a p h s . T e n to s ix t e e n p la n ts w e r e u s e d p e r t r e a t m e n t a n d e a c h e x p e r im e n t r e p e a t e d o n e o r m o r e t im e s . S tra ig h t g r o w t h w a s m e a s u r e d b y o n e o f t w o m e th o d s ; 1. d ir e c t m e a s u r e m e n t o f th e d if­ f e r e n c e b e t w e e n t h e in it ia l a n d f i n a l to ta l le n g t h ; o r 2. m e a s u r e m e n t o f t h e in c r e a se in le n g t h o f a z o n e e x t e n d in g f r o m th e tip to a c h a r c o a l- p e t r o le u m j e lly m ark p la c e d 6 m m . f r o m th e tip f o l lo w in g c h e m ic a l t r e a t m e n t . T h e s e c o n d m e th o d w as p r e f e r r e d s in c e t h e m e a s u r e d g r o w in g r e g io n t h e n c o in c id e d w it h t h e z o n e r e s p o n d in g to t h e g e o t r o p ic s t im u lu s . T h e f ir s t m e t h o d w a s e m p lo y e d o n ly f o r t h e s h o o t s , sin ce th e e lo n g a t io n z o n e e x t e n d s o v e r a c o n s id e r a b le le n g t h . I n t h e c a s e o f c o r n sh o o ts, t w o g r o w t h c e n t e r s a r e in v o lv e d ; t h e c o le o p t ile t ip , a n d t h e rib m e r is t e m b e lo w the a p ic a l m e r is te m . R e su lts I n te r a c tio n o f N P a n d IAA O ver th e range o f 2.86 to 1 1 .4 X 1 0 -7 m o les o f IAA p er liter an d N P con ­ cen tration s of 10- 5 , 1 0 -4 , and 5 X 1 0 ^ 4 M, th ere w a s n o co m p etitiv e in ter­ action b etw een IAA an d N P (F igure 1) in A ven a se ctio n grow th. At high IAA co n cen tration s (5.72 to 1 1 5 X 1 0 -6 M) an d 5 X 1 0 ~ 4 M N P , com p etitive in h ib itio n w as ob served (F igure 2). L ow er co n ce n tr a tio n s o f N P w ere not tested co m p etitiv ely o w in g to th eir in e ffe c tiv e n e ss in in h ib itin g th e geotropic response. T h e in h ib itio n o f pea root section g ro w th ca u se d b y 2.86 to 28.6 X 0.8 10' 0.6 O M NP 0.4 0.2 20 I/S F ig u re 1. I n te r a c tio n in A v e n a c o l e o p t i l e s e ctio n g r o w t h t e s t o f IA A a n d NP. 1/S = 1/IAA c o n cen tra tio n in mg/1. l/G = l/g r o w th in m m /s e c tio n /1 2 hr. P h y s i o l . P l a n t . , 0, 1956 SELECTIVE IN HIBITIO N OF THE GEOTROPIC RESPONSE BY NP 549 o 0.8 0.6 0.4 0.2 0.4 0.6 0.8 I/S Figure 2. In tera ctio n of IAA a n d N P in Aven a co leo ptile section g r o w t h test at high /.4.-I concentrations. 1 /S = 1/I A A co n cen tration in mg/1. l/G = l/g ro \v th in m m /sectio n /1 2 hr. 10 7 M IAA w as not relieved b y 5 X 1 0 -4 M NP. E p in astic effects have been observed in tom ato plants treated w ith 10 7 M NP (H offm ann and Sm ith). Thus N P cannot u n am b igu ou sly be classified as a grow th prom oting su b ­ stance or as a com p etitive in h ib itor of IAA action. It should be pointed out that the com p etitive in hibition data m ay not hold true for sh orter tim e in tervals. A striking exam p le o f this is the 2,6 su b ­ stituted p h e n o x y acetic acids (Osborne et at) w h ich w ere active in extension growth w h en m easu rem ents w ere m ade after short tim e intervals. T im e course of stra ig h t g ro w th a n d geo trop ic curvature The tim e cou rse o f straight grow th and geotropic curvature in control m aterials w as studied in order to select a m inim al incubation period on the linear portion o f the tim e-respon se curve and o f ea sily m easurable m a g n i­ tude. R esults for the several plant m aterials used are su m m arized in Figures 3— 7. In all the plant m aterials tested, grow th is a linear function of tim e for at least four hours. T he geotropic curvature reaches a m axim um at 4 hours (earlier in corn roots), then rem ains constant or som etim es decreases as the bending zon e b ecom es m ature. T h ese results in dicate that under the co n d i­ tions em p loyed , a direct correlation betw een rate o f straight grow th and rate of geotropic curvature exists. P hysiol. P la n t., 9, 195C 550 T E -M A Y TSO U CHING, RO BE R T H . H A M ILTO N AND RO BE R T S. BAN DURSK I 30 0.8 CURVATURE t29 0.6 o cr 32 GROWTH 60 30 X I— 80 29 40 0.4 29 CD 40 30 20 0.2 30 3 0. TIME IN HOURS F ig u re 3. T i m e c o u rs e of g r o w t h a n d g e o t r o p i c c u r v a t u r e of A v e n a c o l e o p t i l e s . Vertical lin es cro ssin g each p o in t rep resen ts + 2 X th e stan d ard error. T h e n u m b er o f p lan ts used is sh o w n fo r ea ch p oin t. CURVATURE 29 29 29 50 47 29 GROWTH 49 29 46 29 X) 40 m m cn 30 o H C on CD om X) 47 m 0 TIME IN HOURS F ig u re 4. P h y s i o l . P l a n t . , 9, 1956 T i m e c o u r s e o f g r o w t h a n d g e o t r o p i c c u r v a t u r e of c o r n ro o ts. SELECTIVE IN HIBITIO N OF THE GEOTROPIC RESPONSE BY NP CURVATURE 551 70 60 48 IN MM 50 42 40 GROWTH GROWTH 30 20 20 TIME IN HOURS F igu re 5. T im e cou rse of g r o w t h a n d g eo trop ic cu rv a tu r e of corn sh oots. 3 CURVATURE 21 60 S a jo IN MM m 2 r44 GROWTH 25 cn c 40 o JO < GROWTH r> 20 c JO 25 m 0 TIME IN HOURS F igu re 6. T im e cours e of g r o w t h an d g e o t r o p ic cu rv a tu r e of pea roots. Physiol- P lant., 9, 1956 552 T E -M A Y TSO U CHING, RO BERT H . H A M ILTO N A ND R O BE R T S. BANDURSK I 50 o m 40 g GROWTH curvature 20 30 m m 20 m TIM E IN HOURS F ig u re 7. T i m e c o u rs e of g r o w t h a n d g e o t r o p i c c u r v a t u r e o f p e a sh o o ts . D is p r o p o r tio n a l in h ib itio n of str a ig h t g r o w t h a n d g e o tr o p ic cu rv a tu r e of s h o o ts a n d ro o ts of corn a n d p e a S traigh t grow th an d geotrop ic cu rvatu re o f sh o o ts a n d roots o f corn and pea w ere d eterm in ed after a 4 h o u r in cu b a tio n fo llo w in g a o n e h o u r pretreatm en t o f th e p lan t m aterial w ith th e v a rio u s co n ce n tr a tio n s o f NP. The exp erim en ta l d ata fo r each p la n t m a teria l tested are c a lcu la ted as percent o f the con trol an d p lotted in F ig u res 8— 11. L o w co n ce n tr a tio n s o f NP, e.g. 1 0 -5 M, in h ib it b oth geotrop ic cu rvatu res an d stra ig h t g ro w th o f shoots 100 80 o GROWTH 60 8 40 CURVATURE u_ F ig u re 8. S t r a i g h t g r o w t h and g e o t r o p i c c u r v a t u r e of N P p re­ t r e a t e d c o r n s h o o t s e x p r e s s e d as p e r c e n t of t h e b u f f e r t r e a te d con­ tro l. 2 20 — v v-4 MOLAR CONCENTRATION P h y s i o l . P l a n t . , 9, 1956 SELECTIVE IN HIBITIO N OF THE GEOTROPIC RESPO NSE BY NP 553 100 g ro w th o CC Iz o o \CURVATURE Figure 9. S tra ig h t g r o w t h an d geotropic cu rv a tu r e of N P p r e ­ treated co rn r o o ts e x p r e s s e d as p e r cent of the bu ffer t r e a te d contro l. I0 ‘ 5 I0 -4 I0 "3 MOLAR CONCENTRATION (Figures 8 and 10) b y ap proxim ately 30 percent. At 10 4 and 10“ 3 M a marked d isp rop ortion ality is m anifested; 10-3 M cau sin g an alm ost total inhibition o f the geotropic curvature of sh oots and on ly a 40 percent in h ib i­ tion of straigh t grow th. A som ew h at sim ilar situation ch aracterizes the curvature and straigh t grow th of root tissue (Figures 9 and 11) except that the d isproportionality b ecom es evident at low er in hibitor concentrations. A 70 to 80 percent in h ib ition o f the tropic response is caused by 10-5 M N P : whereas straight grow th is in h ib ited on ly 20 to 20 percent. 100 80 GROWTH o 40 CURVATURE 20 — tgL F igu re 10. S traigh t g r o w t h and g e o tr o p ic cu rvature of N P p r e ­ t r e a te d p ea s h o o ts exp ressed as p e r cent of the buffer trea te d co n ­ trol. MOLAR CONCENTRATION P h ysio l. P la n t., 9, MUG 554 t e -m a y t s o u c h in g , robert h . Ha m il t o n a nd r o b e r t s. ba n d u rsk i 100 80 GROWTH O 40 20 CURVATURE F ig u re 11. S t r a i g h t g r o w t h a n d geo­ t r o p i c c u r v a t u r e of N P p re-trea ted p e a r o o t s e x p r e s s e d as p e r cent of t h e b u f f e r t r e a t e d co n tro l. TOo MOLAR CONCENTRATION T h e co n cen tration s in d u cin g 50 p er cen t in h ib itio n o f ea c h resp onse for each p la n t organ , as estim a ted g ra p h ica lly , are su m m a rize d in T ab le 1. T h is p ecu liar, “u n c o u p lin g ” o f g row th an d g eo tro p ic cu rv a tu re b y N P occurs in root an d sh o o t tissu es o f b oth m o n o c o ty le d o n o u s an d d icotyled on ou s p lants. P r o p o r tio n a l in h ib itio n of s tr a ig h t g r o w t h a n d g e o t r o p ic c u r v a tu r e of the A v e n a co leo p tile b y N P S in ce A vena co leo p tiles are v ery se n sitiv e to g ro w th regulators, an d their p h y sio lo g ica l b eh a v io r is w e ll k n o w n , ea rlier ex p er im en ts w ere perform ed u sin g in tact A vena coleo p tiles. It w a s fo u n d th a t N P in h ib itio n w a s propor­ tio n a l to the co n cen tration o f th e c h e m ic a l u sed , a n d fo r a sin g le con cen tra­ tion, in h ib itio n in creases w ith in crea sin g tim e o f in cu b a tio n . D ata for a 90 m in u te in cu b ation fo llo w in g a 30 m in u te p retrea tm en t are p lo tted in F igu re 12. T ab le 1. C o n cen tration s in M o f N P and DCIBA req u ired fo r o n e -h a lf in h ib itio n of straigh t grow th and g eo tro p ic cu rvatu re in d iffe r e n t p la n t organ s. M aterial C orn ro o ts ......... A vena co leo p tile Corn ro o ts Corn sh o o ts . . . . P ea ro o ts .......... P ea sh o o ts . . . . P h y sio l. P la n t., 9, 1956 C h em ica l G row th C urvature DCIBA NP NP NP NP NP i X i o —± 1 X 1 0 —4 > 1 X 10 —3 > 1 X 1 0 -3 1X 10-3 > i X i o - 3 i X 10-4 5X10-" 4 X 10—(! l X i o —1 < l X l°~5 2X 10-5 1 SELECTIVE IN HIBITIO N OF THE GEOTROPIC RESPONSE BY NP 555 100 80 o DC Figure 12. S tra ig h t g r o w t h a n d geotropic cu rv a tu r e of N P p r e ­ tre ated A v en a co leo ptiles ex p r e ss e d as per cent of th e b u ff e r tr e a te d control. I2 o o O 60 GROWTH CURVATURE 20 U/L I0 '5 5X10-5 10-4 5X10-4 )0'3 MOLAR CONCENTRATION There is a direct p arallelism betw een in h ib ition of straight grow th and inhibition o f geotropic cu rvature in this tissue. Avena coleop tile tissue thus responds d ifferen tly from the other plant m aterials tested. 120 100 -o 1 8 0 DC GROWTH CURVATURE 20 -M M O LAR Figure 13, CO N C EN TRATIO N Stra ig h t g r o w t h an d g e o tr o p ic cu rv a tu re of DC.IBA p re -tr ea ted corn roo ts e x p r e s s e d as p e r ce nt of the b u ffer tr e a te d control. P h ysio l. P la n t., 9, 1956 556 T E -M A Y TSO U CHING, RO BE R T H. H AM ILTO N AND RO BE R T S. BAN DURSK I P r o p o r ti o n a l in h ib itio n of g r o w t h a n d g e o t r o p ic c u r v a tu r e of corn ro o ts b y D C IB A a n d IAA F o r com p a riso n w ith th e resu lts o f N P -trea tm en t, 2 ,4 -d ich lo ro p h en o x y iso b u ty ric acid (DCIBA) w a s tested for in h ib itio n o f b o th g ro w th a n d geotropic curvature. DCIBA h as b een reported to stim u la te w h e a t root g ro w th (Burstrom , 1951) an d to co m p etitiv ely in h ib it IAA a ctio n in th e A ven a section test (M cRae an d B on n er 1953). DCIBA, th u s, h a s so m e o f th e properties a sso cia ted w ith a n tia u x in s. T h e ex p er im en ta l d ata are p lo tted as p ercen t of the con trol in F igu re 13. A d irect correlation o f stra ig h t g ro w th a n d geotropic cu rvatu re is clea rly ob tain ed . P rop ortio n a l in h ib itio n a p p a ren tly is not a g en eral p rop erty o f an tia u x in s, sin ce B r u m field (1955) o b serv ed a disprop ortion al in h ib itio n o f grow th an d g eotro p ic cu rv a tu re in tim o th y roots fo l­ lo w in g treatm en t w ith a n oth er an tia u x in , 2 ,4 ,6 -tr ich lo r o p h en o x y a ce tic acid. T h e effec ts o f IAA on g row th an d g eo tro p ic cu rv a tu re o f corn roots were exam in ed , an d in co n firm a tio n o f resu lts re ce n tly p resen ted b y B rum field, IAA treatm en t resu lts in p rop ortion al in h ib itio n o f g ro w th a n d geotropic curvature. D is c u s s io n T h e geotrop ic resp on se o f p lan t organ s m a y b e v isu a liz ed as occurin g in three steps: 1. the p ercep tion o f th e stim u lu s; 2. th e tra n sp o rt o f th e stim ulus or a p roduct resu ltin g from th at stim u lu s; a n d 3. the g ro w th resp on se. Either the elon gation zon e or the tip m a y serve as th e p ercep tio n zo n e as show n b y th e ex p erim en ts o f K eeble, N elson a n d S n o w (1929) a n d m ore recently b y A nker (1954). L oss o f geotrop ic se n sitiv ity fo llo w in g d eca p ita tio n is due, ap p aren tly, to rem oval o f th e sou rce o f g ro w th su b sta n ce. T h ere is no general agreem en t as to the m ec h a n ism o f th e g ra v ita tio n a l p ercep tio n or th e trans­ port o f stim u lu s su b seq u en t to th e p ercep tion. It w ill s u ffic e for th e present p u rp oses to con sid er the th e o ry p rop o sed b y W en t a n d C holodn y. These au th ors b eliev e th at gravity a ffe c ts a d isp la cem en t o f g ro w th su b stan ces, and that th e resu ltan t a sy m m e tr ic d istrib u tio n ca u se s d iffe r e n tia l g ro w th o f the u pp er a n d lo w er sid es o f the p lan t organ; th u s b en d in g o ccu rs. A direct correlation b etw een grow th an d g eotro p ic cu rvatu re m a y th u s b e expected and, as a first ap p roxim ation , in h ib itio n o f stra ig h t g ro w th sh o u ld alw ays result in p rop ortion al in h ib itio n o f cu rvatu re. T h is e x p ec ta tio n is experi­ m en ta lly realized in the case ot DCIBA an d IAA in h ib itio n o f root grow th an d cu rvatu re an d is also ap p aren t from th e co rrela tio n b etw een grow th and g eotrop ic cu rvatu re in u n treated con tro l m a teria l. T he selectiv e in h ib itio n P h y s i o l . P l a n t . , 9, 1956 o f geotrop ic cu rv a tu re b y NP is d iffic u lt to SELECTIVE IN HIBITIO N OF TH E GEOTROPIC RESPONSE BY NP 557 explain. It is furth er d iffic u lt to understand the dissim ilarities in b eh aviour of Avena coleop tile tissue, w h ich sh ow s proportional in hibition of straight growth and curvature, and the roots and shoots o f corn and peas, w hich show disproportional in h ib ition . Our present data are in su fficien t to d eter­ mine the m ech an ism o f th e selective tropic in h ib ition by NP. Three p o ssi­ bilities present th em selves assu m in g the valid ity of the W ent-C holodny theory: /. An effect of N P upon the lateral transport o f endogenous auxin; 2. An effect o f N P upon the p erception of the gravitational stim ulus; or 3. An effect o f N P upon on ly that fraction o f grow th associated w ith cu rva­ ture and assu m in g tw o com p onents con trib utin g to grow th. W e believe that exp erim en ts recently reported by Anker cast som e doubt upon the im portan ce o f a lateral redistribution o f au xin as a m echanism for the geotropic b en din g. A nker foun d that the geotropic response o f d eca p i­ tated Avena coleop tiles cou ld be restored b y incubation of the decapitated coleoptile section s in solu tion s con taining IAA, in doleacetonitrile and naphthyl acetic acid. He did not interpret h is results as being at variance w ith the W ent-C holodny theory, and further, did not d eterm ine internal auxin concentrations. It w ou ld appear that redistribution of auxin by coleop tiles im m ersed in a h om ogen ou s solu tion o f IAA is im probable, and therefore an effect o f N P upon internal au xin redistribution w ould not be a lik ely e x ­ planation. An effect o f N P upon the perception m echanism is sim ilarly open to objection. N P h as been reported b y N etien and Conillot lo inhibit the p h o to ­ tropic response. T h ese authors did not present quantitative data to show that the in h ib ition w a s not due sim p ly to an in hibition of grow th by NP. In view of th e results reported here, how ever, it w ould seem that NP m ay inhibit grow th and the p hototropic response d isproportionally. Since the p er­ ception o f the p hotostim u lu s and the perception of the geo-stim ulus probably involves d issim ila r m ech an ism s, it seem s u n lik ely that NP acts to inhibit both sensory m ech an ism s. The p ossib ility w as entertained that straight grow th con sists of tw o co m ­ ponents, and on ly one o f these grow th com p onents is effective in producing curvature. If then N P w ere to inhibit on ly that fraction of grow th norm ally associated w ith curvature, a d isproportional in hibition of straight grow th and curvature w ou ld be observed. T h is exp lan ation appears u nlikely because NP at lO-6 M sig n ifica n tly in h ib its curvature w ith no in hibition of straight growth (Figure 9). We are therefore left w ith ou t a suitable exp lan ation o f the m ode of NP action. Our current concept is sim p ly that NP prevents the local acceleration and/or in h ib ition o f grow th cau sed by gravity. The elucidation o f the m ech a ­ nism of this action rem ains for future studies. P h y s io l. P la n t., 9, 1956 558 T E -M A Y TSO U CHING, RO BERT H . H A M ILTO N AND R O BE R T S. BAN DURSK I S u m m a ry 1. O ver th e tim e in terval for g eotro p ic b e n d in g to rea ch a m axim um , a d irect correlation ex ists b etw een th e rate o f stra ig h t g ro w th a n d geotropic cu rvatu re in u n treated A ven a co leo p tiles a n d th e y o u n g roots a n d sh oots of corn an d pea. 2. A d isp rop ortion al in h ib itio n o f g ro w th an d cu rv a tu re w a s fou n d in n -l-n a p h th y lp h th a la m ic acid (NP) treated roots an d sh o o ts o f corn and pea, but n o d isp ro p o rtio n a lity w a s ob served in N P trea ted A ven a co leo p tiles. 3. Corn roots treated w ith 2 ,4 -d ic h lo r o p h e n o x y iso b u ty r ic a cid sh ow ed a p rop ortion a l in h ib itio n o f straigh t g row th a n d g eo tro p ic cu rvatu re. 4. T he clo se correlation b etw een stra ig h t g ro w th an d g eo tro p ic curvature to be exp ected on the b a sis o f th e W en t-C h o lo d n y th e o ry w a s n ot obtained. R e fe r e n c e s Anker, L.: A co m p a ra tiv e stu d y o n th e r eco v e ry o f th e g e o tr o p ic r e sp o n se o f decapitated A vena c o le o p tile s by in d o le a c e tic acid , in d o le a c e to n itr id e an d n a p h th y la c e tic acid. — K. N ederl. A kad. W eten sh a p . P roc. S ec. C. 5 7 :3 0 4 . 1954. B ru m field , R. T.: T h e in h ib itio n o f cu rvatu res in tim o th y ro o ts b y certa in ch em icals. — Am. J. B ot. 42: 958. 1955. B u rstro m , H.: S tu d ies on g ro w th and m eta b o lism o f ro o ts. VI. T h e rela tiv e grow th action o f d ifferen t iso b u ty r ic acid d eriv a tiv es. — P h y s io l. P la n t. 4 :4 7 0 . 1951. G rigsby, B. H., T so u , T e May, & W ilso n , G. B.: S om e e ffe c ts o f A lan ap (N P) and am izole on g erm in atin g p eas. — P roc. 11th N orth C entral W e e d C on trol C onf. 88. 1954. H o ffm a n n , O. L. & S m ith , A. E.: A n e w grou p o f p la n t g ro w th reg u la to rs. — Science 109: 588. 1949. K eeble, F ., N elso n , M. G. & S n ow , R.: T h e in te rg ra tio n o f p la n t b eh a v io r. I. Separate geo­ tro p ic stim u la tio n s o f tip an d stu m p in ro o ts. — P roc. R oy. S oc. L on d on , Sec. B. 105: 493. 1929. M cRae, D. H., F o ster, R. J. & B on n er, J.: K in etics o f au xin in te r a c tio n . — P la n t P hysiol. 2 8 :3 4 3 . 1953. M cRae, D. H., & B on n er, J.: C h em ical stru ctu re an d a n tia u x in a ctiv ity . — P h y s io l. Plant. 6: 485. 1953. M entzer, C., M olho, D., & P a c h e c o , H.: R ela tio n s en tre la stru ctu re ch em iq u e et l ’inhibitio n d es tro p ism es ch cz les v egetau x. — B ull. S oc. Chirn. B io l. 32: 572. 1950. M entzer, C., & N etien , G.: Sur un p roced e p erm ettan t de trou b ler le g e o tr o p ism e d es racines. — B ull. M ens. S oc. L in n een n e. L yon , 19: 102. 1950. N etien , G., & C on illot, R.: A ction de la cid e n a p h th y l-p h ta la m iq u e su r la germ in ation et les P rem iers stad es de c r o issa n ce d es vd getaux. — B u ll. M ens. S oc. L in n een n e, Lyon, 20: 49. 1951. O sb orn e, D a p h n e, J., B la ck m a n , G. E., N ova, S., S u d zu k i, F ., & P o w e ll, R. G.: T h e p h ysio­ lo g ica l a ctiv ity o f 2,6 -su b stitu ted p h e n o x y a cetic a cid s. — Jou r. E xp . B ot. 6: 392. 1955. W en t, F. W ., & T h im a n n , K. V.: P h y to h o r m o n e s. — M acM illan Co., N. Y. 1937. P hysiol. P la n t., 9, 1956 PHYSIOLOGIA PLANTARUM p u b lis h e d b y t h e S C A N D IN A V IA N S O C IE T Y FOR PLANT P H Y S IO L O G Y is o p e n t o c o n t r ib u t io n s f r o m S c a n d in a v ia n a n d f o r e ig n m e m b e r s o f t h e s o c ie t y . T h e j o u r n a l is is s u e d q u a r t e r ly in n u m b e r s o f a b o u t 1 0 0 p a g e s . T h e s u b s c r ip t io n p r ic e f o r n o n ­ m e m b e r s is 5 0 D a n is h K r o n e r a y e a r . O r d e r s s h o u ld b e p la c e d w it h E j n a r A l u n k s g a a r d , N o r r e g a d e 6, C o p e n h a g e n , D e n m a r k , o r a n y b o o k s e lle r a ll o v e r t h e w o r ld . C o r r e s p o n d e n c e c o n c e r n in g e d it o r ia l m a tt e r s s h o u ld b e a d d r e s s e d to P r o f. H . B u r s tr o m , B o t a n ic a l L a b o r a t o r y , L u n d , Sw eden. Lund 1956. Carl Bloms Boktryckeri A.-B. BIOLOGICAL ACTIVITY OF TETRAZOLE ANALOGUES OF INDOLE-3-ACETIC ACID AND 2 , 4-DICHL0R0PHEN0XYACETIC ACID By R, H. Hamilton and A* K iv ila a n and J. M. McManus P la n t P h y sio lo g y , V ol. 35* No. 1 , 136-140, i 960 B IO L O G IC A L A C T IV IT Y A C ID A N D C rops D iv is io n , OF TETRAZOLE AN A LO G U ES 2 ,4 -D IC H L O R O P H E N O X Y A C E T IC O F IN D O L E -3 -A C E T IC A C I D 2* 3 R. H. H A M IL T O N R esearch A g r ic u l t u r a l R esearch S e r v ic e , U n it e d S tates D epartm ent of A g r ic u l t u r e and A. K IV IL A A N D e p a r t m e n t s of B o t a n y a n d and P l a n t P athology a n d T h e present report is concerned w ith the bio­ logical a ctivity of tw o grow th substance analogues in w hich the tetrazole group has been substituted for the carboxyl group. T h e structure of th ese com ­ pounds, 5- (3 '-in d olem eth yl) tetrazole ( I M T ) and 5(2', 4'-d ich lorop h en oxym eth yl)tetrazole ( 2 ,4 - D T ) is as show n b e lo w : m —N —j r C c J H S tate U n iv e r s it y , E a s t L a n s in g induced by in d o le-3 -a cetic acid ( I A A ) and 2,4-dich lorop h en oxyacetic acid ( 2 ,4 - D ) in a m anner e x ­ pected for a w eak au xin . B y con trast, 2 ,4 -D T does not prom ote g ro w th in the A v en a a ssa y and inhibits both 2 ,4 -D and I A A induced g ro w th . A dditional data su g g est that the in h ib ition of 2 ,4 -D induced g row th by 2 ,4 -D T m ay be p artially ex p la in ed by in­ terferen ce o f 2 ,4 -D uptake by 2 ,4 -D T . a N— N z M u H ^N C -C H V Cl J. M. M cM A N U S C h e m is t r y , M ic h ig a n H Ct A n ex ten siv e program of syn th esis and testin g of tetrazoles has been conducted by H erb st and a sso ­ ciates (2 , 3, 5, 7, 1 5 ). T etrazoles substituted only in the rin g carbon ( R -C N 4H ) are acids h a v in g one d issociable hydrogen. W h en R is alkyl, the d eriva­ tives are sligh tly w eaker acids, w h ile w hen R is arom atic the d erivatives are som ew hat stron ger acids than the corresp ond ing carboxyl com pounds (5 , 11, 1 5 ). H erb st proposed the use of these d erivatives as analogues of b iologically im portant carboxyl com ­ pounds. T h e plant grow th regu latin g a ctiv ity of these com pounds is of in terest sin ce only a few grow th substances w ith ou t a carboxyl group (o r group con­ vertible to a ca rb oxyl) are know n. V eld stra (1 2 , 13) reported a ctivity for several sulfonic, phosphonic, and phosphonous acids. G rowth prom oting a ctiv ity o f som e tetrazole d erivatives w as reported by V an de W esterin g h and V eld stra ( 1 1 ) , subsequent to com ­ p letin g the present studies. A brief prelim inary re­ port of the present w ork has appeared ( 4 ) . It w ill be show n that IM T prom otes elongation of A v e n a coleoptile sections and antagon izes grow th 1 Received June 12, 1959. 2 Journal Article 2392 from the Michigan Agricultural Experiment Station. 3 Supported in part by the National Science Founda­ tion. ethods IM T and 2 ,4 -D T w ere sy n th esized by reaction of the n itrile w ith alum inum azid e and sodium azide respectively. T h e d etails o f sy n th esis and character­ ization are to appear elsew h ere ( 7 ) . T h e purity of IM T w as exam in ed by ch rom atograp hy o f 1 m g quantities of IM T on W h atm an N o . 1 paper u sing isopropyl a lc o h o l/a m m o n ia /w a te r ( 8 : 1 : 1 ) ; pyri­ d in e/a m m o n ia ( 4 : 1 ) ; and 77 % eth anol as ascending solvents. In the last tw o so lv en ts IM T ran slightly above I A A and w hen sprayed w ith E h rlich ’s reagent (1 % />-dim ethylam inobenzaIdehyde plus 8 . 5 % conc. HC1 in 95 % eth a n o l) g a v e a purple spot changing to yellow . N o I A A or in d o le-3 -a ceto n itrile w as de­ tected as contam inant of the IM T . S in ce, under these conditions, 0.2 Mg' of I A A or the n itr ile could have been detected, these com pounds could n ot account for the grow th p rom otin g a ctiv ity of IM T . V icto ry oats w ere husked, soaked in tap w ater, and placed em bryo up on the ed ges of p lastic blocks covered w ith m o ist filter paper. T h e seed lin g s w ere exposed to con tinu ous lo w in ten sity red lig h t at 25° C and 95 % hum idity for about 60 hours. C oleoptiles 25 to 35 mm in length w ere selected and one 6.5 mm section w as cut 2 mm from the tip. In som e ex p eri­ m ents, the prim ary lea f w a s rem oved, but in m ost exp erim en ts it w as not. T h e sectio n s w ere floated in P etri dishes on 10 m l of 0.01 M K H 2P O , buffer pH 4.5 co n ta in in g 2 % sucrose. C oncentrated stock solu tions o f the grow th reg u la tin g acids w ere pre­ pared by d isso lv in g th e acid in a slig h t m olar excess of alkali. G row th w as m easured at 5, 10, and 20 hours on *he sam e lo t o f sectio n s b y m ean s o f a' bi­ nocular m icro sco p e w ith an ocular m icrom eter. T h e 5 and 10 hour m easu rem ents w ere m ade as rapidly as possib le u sin g illum in ation from a 7.5 w a tt red safeligh t. E x p erim en ts in dicated that sectio n s m easured 136 H A M IL T O N ET A L— TETRAZOLE A N A L O G U E S OF at 5 and 10 hours had not elongated sign ifican tly less at 20 hours than sections m easured after 20 hours only. All data represent the average of at least tw o or three experim ents u sing about 25 sections per treatm ent dish. Seeds of bean ( Phaseolus vulgaris, var. W a x Pencil P o d ) w ere planted in soil in the greenhouse and used at the prim ary leaf stage ju st before the terminal bud unfolded. S olutions of grow th sub­ stances used for treatm ent w ere in 95 % ethanol plus 0,1 % T w een 20, and treatm ents m ade by application of a 10 drop to one prim ary leaf. R IAA AND 2,4-D 137 4 .0 30 2.0 A = I0 “ 4 M J M TETRAZOLE A. » 5 * 1 0 -® M IM TETR A ZO LE p ■ 10“ * M IM TETR AZOLE ■ * NO IM TETRAZOLE esults 5 -(3 '-I n d o l e m e t h y l ) T et r a zo l e: T he grow th rate of A ven a sections used in these studies w as not a linear function of tim e but tended to decrease sligh t­ ly at the longer incubation tim es. T h e buffer used, pH changes during incubation, su ccessive m easure­ ment, or the particular lot of seed used did not ac­ count for the non-linear grow th. T h e tim e course (fig 1) represents the average of a number o f exp eri­ ments. A lthough the deviations from linearity w ere not large, they w ere consistent. A low grow th rate for the control sections, and to som e extent for the 2,4-D treated sections, w as noted during the first 5 hours. O nly grow th m easurem ents obtained after a 10 hour incubation are here presented since these 1.4 x 10' 2 .6 X 1 0 " 2 .8 * tO 5 .7 x I 0 " 7 IAA MOLES PER LITER - 30 A = 10"* M IM TETRAZOLE A = 5 x t 0 '» M IM TETR A ZO LE p = l® -» M IM TETR AZOLE 7.0 ■ - NO IM TETRAZOLE 6.0 1 .2 5 x 2.8 X I06M IAA 2 .2 5 X I0 6M j j et fci 4.0 S 3.0 5 CONTROI I05 M 2 ,4 -D T 20 TIME IN HOURS Time c o u r s e for growth of Avena coleoptile sections when floated in solutions containing IA A , 2,4-D, 2,4-DT, IM T or no growth substance. Vertical lines in figures represent dr 2 X standard error. F i g . 1. I0 "7 10" * F ig . 2. Growth of Avena coleoptile sections after 10 hrs in three concentrations of IM T alone, and in com­ bination with various concentrations of IAA. 10* M IMT S 2.0 25 X F ig . 3. Growth of Avena. coleoptile sections after 10 hrs in three concentrations of IM T alone, and in com­ bination with various concentrations of 2,4-D. CD ** 2 2 , 4 - 0 MOLES PER LITER 24-D * 5.0 I0 "T data are representative but less subject to error and non-linearity than the 5 or 20 hour m easurem ents. A s is shown in figures 1, 2, and 3, IM T promotes elongation of A v e n a coleoptile sections. T hus 1 0 - 4 M IM T is equivalent as a grow th promoter to 2.8 X 10-7 M IA A ( fig 2 ) , or to 2.25 X 10- e M 2,4-D ( fig 3 ) . B y contrast, the effect of IM T in com bina­ tion w ith IA A ( fig 2 ) , or 2,4-D ( fig 3 ) m ay be stim u ­ latory, inhibitory or nil depending on the concentra­ tion of 2,4-D or IA A . T h ese results are interpretable in terms of a weak grow th substance com peting w ith a stronger one. A t higher concentrations of IA A or 2,4-D , the less active IM T could occupy a number of binding sites thus blocking out the m ore active IA A or 2,4-D and resulting in inhibition of the IA A or 2,4-D induced growth. PLANT 138 T able E ffect I of 2,4-D T nduced E 1 IA A or l o n g a t io n of on P H Y S IO L O G Y 2,4-D .A v e n a C o l e o p t i l e S e c t io n s A u x in CON c m olar IAA* 1.4 2.8 5.7 2.8 2,4-D* 1.25 2.25 4.50 2.25 0 X X X X 0 X X X X 0** 10~7 1 0 -7 10“ 7 1 0 -6 1 0 -7 10-7 10~7 10“ 6 1.54 2.74 3.56 3.89 4.32 1.08 1.09 1.50 2.07 3.26 ± 0.054 ± 0.010 ± 0.15 ± 0.15 ± 0 .1 1 ± 0.052 ± 0.058 ± 0.064 ± 0.10 ± 0.14 % I N H IBITIO N 2,4-DT M o l a r c o x c lO "5 5 X 1 0 - 5 lO - 4 27 1 7 12 2 24 24 27 29 15 38 21 17 19 11 44 50 49 56 37 51 35 35 30 27 67 69 71 71 51 * Average of three experiments with 2,4-D and three with IA A . Section length measured after 10 hrs ** Growth in millimeters ± 2 x standard error 5 -(2 ', 4 '-D ic h l o r o p h e n o x y m e t h y l ) T etrazole: T he data of table I show that 2 , 4 - D T did not pro­ m ote elongation of A v e n a coleoptile section s at any of the concentrations tested and w as, in fact, m ea s­ urably inhibitory to grow th at concentrations greater than 1 0 _D M. T h e interactions of 2 ,4 -D T w ith 2,4- D and I A A w ere rather co m p lex ; th e n atu re o f the in teractions is m ost clearly sh ow n by a double recipro­ cal plot of the data. T h u s 2 ,4 -D T acted as a weak and n on -com p etitive in hibitor of I A A induced grow th ( f i g 4 ) , but the in teraction of 2 ,4 -D T and 2,4-D ( f i g 5) is quite different, and a co m p etitiv e com ponent is noted. T h e nature of the in tera ctio n s is also shown in a less strik in g m anner in table I. T h u s, the slight inhibition by 2 ,4 -D T is scarcely in fluenced by IA A concentration. O n the other hand, 2 ,4 -D T inhibits 2 .4-D induced gro w th to a g reater ex ten t and the h ig h est 2 .4 -D con centration tends to overcom e this inhibition. A p p a r e n t I n h i b i t i o n o f 2 ,4 -D A b s o r p t i o n b y 5- ( 2 , 4 - D i c h l o r o p h e n o x y m e t h y l ) T e t r a z o l e : An exp erim en t w as d esign ed to test th e h yp oth esis that 2 .4 -D T m igh t a n ta g o n ize the ab sorption o f 2 ,4 -D by a plant tissue. F o r this exp erim en t, th e prim ary leaf of g re en h o u se-g ro w n bean plants w a s used w ith either separate or coin cid en t application o f 2 ,4 -D and 2,4-D T . T he treatm ents (s e e f ig 6 ) w ere as fo llo w s: 1) con­ trol, no treatm ent, 2 ) 2 ,4 -D T alone, 3 ) 2 ,4 -D alone, 4 ) , 5 ) , 6 ) 2 ,4 -D T and 2 ,4 -D both applied and to d if­ ferent areas of the leaf, 7 ) 2 ,4 -D and 2 ,4 -D T applied coincidently. A fter 24 hours the treated leaf was rem oved, and the curvature o f the stem measured; A s a con ven ien t m easure of form a tiv e response, the first trifo lia te leaf w a s rem oved and w eig h ed after tw o w eeks. It is apparent from a typical experim ent 3 .0 A = I 0 '4 M 2 , 4 - D A TETRAZOLE = 5 x 1 0 " 2, 4- D TETRAZOLE □ = 10 " 9M 2,4-D TETRAZOLE a = I0"4 M 2 ,4 - D TETRAZOLE 0.8 a ■ = NO 2 , 4 - D TETRAZOLE = 5 * 1CT5 M 2 ,4 -D TETRAZOLE a = 1 0 M 2 ,4 -D TETRAZOLE ■ = NO 2 ,4 - D TETRAZOLE 2.0 0.2 0 .3 6 1.75 3.57 IAA LITERS /p.M 7.15 0 0 ,4 4 2 .2 2 4 .4 5 8.00 2 , 4 - D LITERS /JJ.M F i g . 4. Reciprocal of growth of Avena coleoptile sections after 10 hrs in presence or absence of 2,4-D T plotted as a function of the reciprocal of various concentrations of IA A . Fig. 5. Reciprocal of growth of Avena coleoptile sections after 10 hrs in presence or absence of 2,4-D T plotted as a function of the reciprocal of various concentrations of 2,4-D. H A M IL T O N ET A L TETRAZOLE A N A L O G U E S OF Location of treatment Stem Trifoliate 2 ,4 - D 2,4-D T ©=2,4-D leaf 690). The pK estim a ted from th e u l t r a v i o le t s h i f t i s 7*0. The product sub lim es upon h ea tin g in vacuo (90° @ 0 .0 5 mm Hg). T reat­ ment o f a y e llo w s o lu t io n o f th e product in 50 $ eth a n o l w ith zin c and 1 drop o f 6 N HC1 y i e l d s a c o lo r le s s s o lu t io n . N it r a t io n o f corn sw eet compound About 50 mg o f CSS was d is s o lv e d in 1 .5 ml o f aceton e and t r e a te d w ith 0 .1 m lo f concentrated HNO^. There was an immediate form ation o f a b row n ish -red c o lo r so th e s o lu t io n was d ilu te d w ith 4 ml o f d i s t i l l e d w ater and c o o le d . The orange c r y s t a ls ob tain ed were r e c r y s t a liz e d from aqueous e th a n o l, but had no d e f i n i t e m.p. The product was sublim ed in vacuo (120° @ 0 .0 5 mm Hg) and y ello w c r y s t a ls c o lle c t e d on th e co ld f in g e r , le a v in g a red r e s id u e . in g p o in t . The y e llo w product had no d e f in i t e m elt­ The u l t r a v i o l e t maxima, minima and th e s p e c tr a l s h i f t w ith pH were i d e n t i c a l t o th e product ob tain ed upon t r e a tin g w ith n itr o u s a c id . P r ep a ra tio n o f an azo dye The p r ep a r a tio n was a f t e r th a t o f Tedder and Theaker ( 2 9 ) on a 50 mg s c a le . When th e diazonium s a l t was coupled to r e s o r c in o l, an imme­ d ia t e d eep -red c o lo r was ob served . The azo dye was r e c r y s t a liz e d from aqueous m ethanol, y ie ld in g about 16 mg o f orange c r y s t a l s , m.p. 221 2 3 1 °. An a n a ly s is fo r C, H and N in d ic a te d th e product was impure. Paper chromatography in ethanol-ammo n ia-w ater ( l O s l s l ) , isopropanol-am m onia-w ater ( l O s l s l ) , and n -bu tan ol-am m onia-w ater-ethan ol (4 4 sls 2 0 s2 0 ) r ev ea led a contam inante g iv in g brow nish-pink c o lo r when sprayed w ith 0 .0 1 N NaOH, and running ju s t above th e red dye. The Rf o f th e dye was r e s p e c t iv e ly -9 8 0*50, 0 .2 8 , and. 0.37* The dye was r e c r y s ta le d from a c e t o n e - lig r o in , but when chromatographed on paper was s t i l l found to be impure. The pK o f th e y e llo w -r e d in d ic a t o r - lik e change o f th e dye was about 8.5* O xidatio n o f corn sw eet compound w ith n eu tr a l permanganate The permanganate rea g en t c o n s is te d o f 0 .0 5 M KMnOi* co n ta in in g 5$ MgSO/j, (30)* a c e to n e . About 0 .1 mM (2 1 .1 mg) o f CSS was d is s o lv e d in 1 ml o f The permanganate was added in 0 .0 5 ml p o r tio n s (0 .0 5 mM) and upon l o s s o f th e p u rp le c o lo r , another a liq u o t was added. I t was n e c e ssa r y to c e n tr ifu g e down th e Mn02 to observe th e c o lo r change. A pproxim ately 5 e q u iv a le n ts o f permanganate were consumed. permanganate was decomposed w ith ammonium o x a la te . The e x c e ss The s o lu t io n was a c i d i f i e d w ith 1 N H2 SO4 u n t i l a l l th e Mn02 went in to s o lu t io n , ex­ t r a c te d w ith 2 volumes o f e th e r , and th e combined eth er f r a c tio n d ried over anhydrous Na2 S04 a t 1 ° . 3 ml o f 1 N NH^OH. The decanted eth er la y e r was shaken w ith A 0 .2 ml sample was chromatographed on paper in ethanol-am m onia-w ater ( 8 0 s 4 s l6 ) . S ix u l t r a v i o l e t absorbing or f lu o r ­ e s c e n t sp o ts were n o ted , none o f which gave a FeCl^ c o lo r . Two a c id s (Rf 0 .1 4 , 0 .2 3 ) , not m atching any o f th e u lt r a v i o l e t s p o ts , were found upon sp ra y in g w ith bromphenol b lu e ( 0 .5 mg per ml p lu s 2 mg/ml c i t r i c a c id ). The a c id , Rf 0 .2 3 , was rechromatographed in 80$ phenol and had an Rf in th e s e two s o lv e n ts v ery c lo s e to g ly c o lic a c id . The oth er a c id had an Rf o f 0 .1 8 in t h is l a t t e r s o lv e n t and an Rf o f 0 .1 6 when chromato­ graphed in n -b u ta n o l-fo rm ic a cid -w a ter ( 1 0 s 2 s l5 ). -9 9 Acknowledgments We w ish to thank Dr. E. E. Smissman o f th e U n iv e r s ity o f W isconsin fo r a s y n th e tic sample o f 6-m ethoxybenzoxazolinone, Dr. Grant Smith o f th e Dow Chemical Company fo r th e therm al degradation a n a ly s is , and Dr. James S h o o lery o f Varian A s s o c ia te s fo r th e n u clear m agnetic resonance s p e c tr a . R eferences 1 . S u h adolnik, R. J. Ph.D. T h esis, P enn sylvan ia S ta te U n iv e r s ity (1 9 5 6 ). 2 . 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