BIOLOGIA PLANTARU~V[ (PRAHA)

1 (1) : 54--62, 1959

A Study of Metabolism of Exogenous Tryptophane and

fl-Indoleaeetie Acid in Extirpated Wheat Embryos

MALAN K U T ~ E K , KV]~TA ROKOSOVA, RUDOLF I~ETOVSK~

Department of Plant Physiology, Instituto of Biology, Czechoslovak Academy of Scionces
and Department of Chemistry of the School of Agriculture, Praha
Received March 12, 1958

Souhrn

Pro objasn6ni lAtkov:~ch p~em6n tryptofanu a kyseliny fl-indolyloctov6 byly

p~e~ivajicim ombryim p~enice v Knopov6 roztoku p~ids bud tryptofan
nebo kyselina fl-indolyloctovA v koncentraci 3 . l0 -2 M. Po 24 hodinAch byly z em-
bryi rychle p~ipraveny alkoholick6 v~luhy, kter6 byly chromatografieky nebo
elektroforoticky analysovhny. Krom6 chromatografick:~ch soustav a detek6nich
6inidel, obvykl~ch p~i analyso indolov~ch slou6enin, byly pou~ity n6kter6 m6n5
zngLm6 syst6my, kter6 se osv6d6ily p~i analyse nov6 objeven6ho indolov6ho deri-
vs rostlin 6cledi B r a s s i c a c e a e - - askorbigenu. Jsou to soustavy octan butylnat:~ na-
sycen~ vodou, tetrachlormetan~-kyselinaoctov~ (10O : 2), formaldehydov6 detek6ni
6inidlo (40~o formaldehyd--konc. HCl--voda 1 : 1 : 2). Paplrovs elektroforesa byla
provg~d6na ve M/15 fosfs pufru pH 6.6 p~i 300 V po dobu 4 a~ 5 hodin.
Hlavni pozornost byla v6novs dflkazu hydrofilnich komplexfl dod~van~ch 1Atek.
V embryich p~e~iva.jicich v roztoclch kyseliny fi-indolyloctov6 byla krom6 jin:~eh
ir~dolov~ch derivAtfi zji~t6na p~itomnost kyseliny fl-indolylacetylasparagov6. Jeji
identita byla prokAzs podle R F v 5 rflzn:~ch soustav~eh barevn:~mi reakeemi
a chromatografick~m dflkazem jejich slo~ek po alkalick6 hydrolyse (6 N Ba(OH)~
po 30 rain. p~i 100~ C). V embryich p~e~ivajicich v tryptofanu byla zji~t6na ls
obdobn~ch chromatografick~ch vlastnosti jako kyselina indolylacetylasparagovA,
kters pak vhak po hydrolyse poskytovala tryptofan. P~edpoklAds se, ~e jde o malo-
nyltryptofan. Krom6 toho byl v jednom pokusu pozorov~n vznik ls chromato-
graficky obdobn6 askorbigenu. Uveden~ prAce potvrzuje odli~n:~miprost~'edky a n a
jin6m biologick6m materiMu sou6asn6 v~sledky Andreaeho a Gooda.

Summary
The m e t a b o l i s m of exogenous t r y p t o p h a n e ( 3 . 1 0 - 2 M ) a n d fl-indoleacetie
acid ( 3 . 1 0 - 2 M ) in e x t i r p a t e d wheat e m b r y o s was studied c h r o m a t o g r a p h i c a l l y
and electrophoretically.
I t was established t h a t the ex t er n al application of fl-indoleacetic acid in
w h e a t e m b r y o s l e a d s t o t h e f o r m a t i o n o f its c o m p l e x , i n d o l e a c e t y l a s p a r t i c
acid. C h r o m a t o g r a p h i c a l d e m o n s t r a t i o n o f t h e c o m p o n e n t s o f t h e c o m p l e x
was m a d e following alkaline hydrolysis.
A c o m p l e x c o m p o u n d also arises f r o m e x o g e n o u s t r y p t o p h a n e , w h i c h
a g a i n frees t r y p t o p h a n e f o l l o w i n g a l k a l i n e h y d r o l y s i s . T h e a s s u m p t i o n h a s
b e e n p u t f o r w a r d t h a t t h i s is m a l o n y l t r y p t o p h a n e , r e p o r t e d b y A n d r e a e

54
 METABOLISM OF EXOGENOUS T RYP T OP HANE ACID 55

and Good ( D A N N E N B U R G and LIVERMAN 1957). The isolated finding of a further

substance may be mentioned, which possesses similar chromatographic pro-
perties to those of the indole complex--ascorbigen. For reasons so far un
known, this substance has not been isolated again in wheat plants.

Introduetion

Many authors ( S K o o G 1937, W I L D M A N , FERRI and BONNER 1947, WILD-

MAn and MUIR 1949, GORDOn and NIEVA 1949, ABRAMS 1953, HEMBERG
1955, PILET 1956) have found that tryptophane is often partially metabolized
to growth substances in plant tissues. The metabolic processes involved in
this conversion have not yet been elucidated and have formed the subject
of many studies (LARSEN 1951, GORDOn 1954). At the end of last year DANNEN-
BBURO and LIVERMAN (1957) published a detailed study of the products of
metabolism of tryptophane-2-C 14 in tissue slices of the water melon and oat
coleoptiles.
It can be assumed that, following the entry of indoleacetic acid into plant
tissue, part of it will be decompbsed b y oxidase (TAXG and BONNER 1947,
MACLACHLANand WAYGOOD 1956, RAY 1956). A further part of the heteroauxin
is bound to protein (WILDMANand GORDOn 1942, WILDMAN and BONNER
1947, SIEGEL and GALSTON 1953). More recent work by Andreae et al. has
shown that exogenous indoleacetie acid forms a complex compound, indole-
acetylaspartic acid (ANDREAE and GOOD 1955, GOOD, ANDREAE and VAN
YSSELSTEIN 1956, ANDREAE and VAN YSSELSTEIN 1956).
In the present study, we attempted to verify current knowledge of the
metabolism of exogenous tryptophane and indoleacetic acid in wheat seedlings,
using a somewhat different method. We were particulary interested in evidence
of hydrophilic complexes of indole in the plant tissues.

Methods

Cultivation of p l a n t s : W h e a t grains (Chlumecks 12. 1956 harvest) were disinfected for a q u a r t e r

of an h o u r in 96O/o ethanol a n d rinsed several times in boiled distilled water. The grains were
g e r m i n a t e d in Petri dishes on roils of filter p a p e r moistened w i t h K n o p ' s s o l u t i o n - - Z A z v o r k a ' s
modification (PETRff a n d I~ETOVSK~ 1956). The Petri dishes wero placed in a glass c h a m b e r
a n d exposed to the n o r m a l a l t e r n a t i o n of d a y a n d night. The average t e m p e r a t u r e was 25 ~ C.
The grains g e r m i n a t e d on the second day. W h e n the average length of the coleoptiles w a s
5 ram. the e m b r y o s were extirpated.
Survival of the e m b r y o s in indole solutions: The e x t i r p a t e d e m b r y o s were kept in K n o p ' s
solution w i t h 3-10-2M t r y p t o p h a n e or 3.10-~M fl-indoleacetic acid. F o r 200 e m b r y o s 20 ml. of
n u t r i e n t solution was used. I n the control e x p e r i m e n t the e m b r y o s were placed in K n o p ' s solu-
tion. The Petri dishes were r e t u r n e d to the glass chambers. After a f u r t h e r 24 h o u r s the e m b r y o s
were r e m o v e d f r o m the solution a n d p r e p a r e d for examination.
P r e p a r a t i o n of extracts: The e m b r y o s were t h o r o u g h l y rinsed w i t h distilled w a t e r a n d dried
of external water. T h e y were t h e n weighed a n d g r o u n d in a m o r t a r w i t h a small a m o u n t of
silica sand. I n the m a j o r i t y of e x p e r i m e n t s a n alcohol e x t r a c t was p r e p a r e d (VALENTA, KUT./~EK
a n d ICHA 1956) - - one p a r t b y weight of the p l a n t material w a s m a d e u p to a total volume
of three p a r t s w i t h 96~o ethanol. The suspension was left in a refrigerator at a t e m p e r a t u r e
of a b o u t + 4 ~ C for two h o u r s before centrifuging. I n some e x p e r i m e n t s sap pressed f r o m the
p l a n t s w a s s h a k e n u p with twice the volume of ethyl acetate.
56 M. K U T / ~ E K , K . R O K O S O V A , R. ] ~ E T O V S K Y

C h r o m a t o g r a p h i c s e p a r a t i o n of indoles a n d d e t e c t i o n of e h r o m a t o g r a m s . C h r o m a t o g r a p h y
w a s carried o u t in a descending, or e x c e p t i o n a l l y in a n a s c e n d i n g a r r a n g e m e n t in t h e following
systems

$1 - - butyl acetate saturated with water

S2 - - n - b u t a n o l - - g l a c i a l acetic a c i d - - w a t e r (4 : 1 : 5)
SS - - c a r b o n t e t r a c h l o r i d e - - g l a c i a l acetic acid (100 : 2)
S4 - - i s o p r o p a n o l - - a m m o n i a (d ~ 0 . 8 9 5 1 ) - - w a t e r (10 : 1 : l)
S5 - - i s o p r o p a n o l - - 8 N - - N t t S (8 : 2)
$5 - - i s o p r o p a n o l c o n t a i n i n g 1.25~o acetic acid--20~ a q u e o u s solution of a m m o n i u m ace-
t a t e (8 : 2)
D e t e c t i o n w a s carried o u t w i t h a) a f o r m a l d e h y d e r e a g e n t (1 v o l u m e 40~o f o r m a l d e h y d e
m i x e d w i t h 1 v o l u m e of c o n c e n t r a t e d hydrochloric acid a n d 2 v o l u m e s of water). C h r o m a t o -
g r a m s were dried a t 70 ~ C. W i t h t h i s r e a g e n t only d e r i v a t i v e s of indole c a r r y i n g a s u b s t i t u e n t
w i t h a m e t h y l e n e g r o u p in t h e a - p o s i t i o n to t h e indole ring in position 3 of t h e indole
n u c l e u s give a positive reaction, p r o d u c i n g s p o t s w i t h yellow or o r a n g e fluorescence in ultra-
violet light; b) Salkowski's r e a g e n t (50 v o l u m e s 50/0 perchlorie acid m i x e d w i t h 1 v o l u m e 0-05
M - - F e C l a ; c) E h r l i c h ' s r e a g e n t (2~o s o l u t i o n of p - d i m e t h y l a m i n o b e n z a l d e h y d e in a m i x t u r e of
20 m l . c o n c e n t r a t e d h y d r o c h l o r i c acid a n d 80 ml. 960/0 ethanol); d) 1~o s o l u t i o n of c i n n a m i c
a l d e h y d e in m e t h a n o l ; e) 0.1~ s o l u t i o n of n i n h y d r i n in n - b u t a n o l ; f) 0.05~o s o l u t i o n of B r o m o -
cresol G r e e n in 960/0 e t h a n o l ; t h e p H is a d j u s t e d to 7.
P a p e r electrophoresis of t h e indole d e r i v a t i v e s w a s carried o u t b y t h e m e t h o d of ULLMAI~
a n d LIEBL (private c o m m u n i c a t i o n ) in M/15 p h o s p h a t ~ buffer of p H 6-6 a t 300 V for a period
of 4 to 5 h o u r s .
Alkaline h y d r o l y s i s of t h e c o m p l e x e s w a s carried o u t in 6 N - B a ( 0 H ) 2 on a boiling w a t e r b a t h
for 30 m i n . (AND]~EAE a n d GOOD 1955).

ResuLts

A. Experiments with fl-indoleacetic acid.

In alcohol extracts of embryos kept in 3.10-2M solutions of fl-indoleaeetic
acid the following spots were found on chromatograms obtained in system S 1
and detected with the formaldehyde reagent:
a) fl-indoleaeetic acid, I~F 0"86 (spot I).
b) a spot of I~F 0"71 showing brownish-yellow fluorescence (spot II),
e) a spot of I~F 0-05 showing orange-pink fluorescence (spot III).
On some chromatograms a yellow fluorescence, which we consider be due
to tryptophane was present at the start (spot IV). When compared with the
experimental embryos, extracts from the control group, without indoleacetie
acid, sometimes showed a not very intense spot of #-indoleacetic acid (spot I),
always an intense spot II of ~:~F 0.71, sometimes a weak spot IV at the
start. Spot III of RF 0.05 was never present.
We attempted to compare spot II with a synthetic sample of fi-indoleaeet-
amide. The RF values of spots of indoleacetamide were, however, clearly distinct
in systems S 1 and S 2 from the RF values of spot II (table I). Similarly, the
colouring of spot II with formaldehyde, Salkowski's reagent and partially
Ehrlich's reagent was different when compared with the spot of indoleacetamide
(table II). When the sap expressed from the embryos is extracted with ethyl
9cetate the compound responsible for spot II readily passes into the solvent.
The orange-pink fluorescent spot III had the same RF values in systems
$1, $2, $4, $5, and $6 as authentic fl-indoleacetylaspartic acid (table I). Final
 METABOLISM OF EXOGENOUS T R Y P T O P H A N E ACID 57

F F F ; F ; D C

O f

H
O
/

c ~ c

Fig. 1. Fig. 2.
Fig. I. Chromatogram of indole derivatives from extracts of' wheat seedlings kept in 3.10-2M
indoleacetic acid (2), 3.10-~M tryptophane (3) and from control seedlings (1). (4.5)
standards. Detection was carried out with formaldehyde reagent. Spots: A fl-indole-
butyric acid (yellow- 13rown fluorescence) B /3-indolcacetic acid (yellow fluorescence---
spot I); C tryptophane (yellow fluorescence spot IV); D malonyltryptophanc (orange-
pink fluorescence - - spot V); E unknown substance similar to ascorbigen (orange fluor-
escence - - spot VI); F unkown substance (brown- yellow f l u o r e s c e n c e - spot II); (i in-
doleacetylaspartic acid (orange-pink fluorescence spot III); H, I unknown substances
(orange fluorescence); J unknown substances (green fluorescence).
F~g. 2. Eleetrophoreogram of indole derivatives from extracts of wheat seedlings kept in 3 . 10-eM
tryptophane (3--5) and from control seedlings (1.2) . (6--9) standards. Elcetrophoretie
division of substances was carried out in phosphate buffer p H 6-68 (cone. M/15), tension
300 V, time of division 4 hours, pontetial declivity 10 v/1 era.
Detection was carried out by formaldehyde reagent. Spot's: A fl-indolebutyrie acid; B indole-
p ropionie acid; E indoleacetamide; D aseorbigen; E indoleaeetylaspartie acid; F tryptophane;
G /~-indoleaeetic acid; H malonyltryptophane.
58 M. K U T J ~ E K , K. ROKOSOV~, R. I~ETOVSK~

Table 1. R F Values of Indoleacetylaspartic Acid (IAA). Indolelacetamide (IAM) and Spot I [

in Some Solvents

System R F IAA R F IAM R F Spot I I

S1 0.05 0.64 0.71

S~ 0.79 0.80 0.82
St 0.09 0.92 0.91
$5 0'07 0.79 0.89
$6 0.27 0.92 0.95

proof of the identity of this spot as indoleacetylaspartic acid was obtained

following elution of the spots and their alkaline hydrolysis. To establish the
presence of fl-indoleacetie acid, spot III was eluted from the chromatogram
in system S~ (RF 0.05), in which the I~F of free fl-indoleacetic acid is 0-86;
chromatography of hydrolysates in system Sa demonstrated the presence
of fl-indoleacetic acid.
The presence of aspartic acid in hydrolysates of spot III was confirmed
following elution of this spot from a ehromatogram run in system S2 (RF 0.79),
hydrolysis, and repeated chromatography.

B. Experiments with tryptophane

On chromatograms of alcohol extracts of embryos kept in 3"10-~M solu-
tions of tryptophane the following spots were always obtained in system S1
after detection with the formaldehyde reagent:
a) a spot of l~s 0"71 showing intense brownish-yellow fluorescence (spot II)
b) a spot of RF 0.08 showing orange-pink fluorescence (spot V)
c) a yellow fluorescent spot of tryptophane at the start (spot IV).
In an experiment with a series of nutrient solutions containing graded
concentrations of tryptophane (3-10-21~r 10-2M, 10-4M, 10-6M) the general
pattern of the spots did not change, only spots IV and V decreased in intensity.
The chromatographic patterns shown by embryos of the control group
without tryptophane have already been described in the foregoing section.

Table 2. Colouring of Spots of Indoleaeetylaspartic Acid (IAA), Indoleacetamide (IAM) and

Spot I I with Some Reagents

Reagent IAA IAM Spot I I

Formaldehyde daylight Y OB B
UV radiation OP O BY
Salkowski's Y R RB
Ehrlich's V YB Y, sometimes YB
Cinnamic aldehyde 0 Y Y

Key: R - - r e d , V--violet, B--brown, O--orange, P - - p i n k , Y--yellow

 METABOLISM OF EXOGENOUS TRYPTOPHANE ACID 59

In the first experiment with tryptophane a further clear spot of RF 0.52,

showing orange fluorescence (spot VI) was found in the chromatogram of
extracts from the embryos. The RF of this spot in systems S 1 and S2 agreed
with the ]~F value recorded for ascorbigen. Similarly, the orange fluorescence
obtained after detection with the formaldehyde reagent and the blue colour
with Ehrlich's reagent are analogous to the reactions of ascorbigen (P~o-
CHXZKA 1957). In the course of further experiments it was not, however,
possible to confirm the presence of this substance; nor did ascorbigen occur
in any of the control experiments.
The substance responsible for spot V shows chromatographic properties
similar to those of indoleacetylaspartic acid. Its Rs was practically the same
as that of indoleacetylaspartic acid in systems S 1, $2, $5, and $6 (table I).
The electrophoretic mobility of the substance producing spot V was also the
same as that of indoleacctylaspartic acid. On extraction of the expressed
sap of the embryos with ethyl acetate only a small part of this substance
passes into the solvent. According to a personal communication from Dr.
Andreae a complex compound, malonyltryptophane is formed in plant tissues
from exogenous tryptophane. The chromatographic properties of malonyl-
tryptophane as described b y him, are similar to those of indoleacetylaspartic
acid. Hydrolysis of the complex with barium hydroxide was carried out in
eluates of the zone cut from the electrophoreogram, where spot V is sufficiently
separated from tryptophane. No indoleacetic acid was present in the hydroly-
sate. Detection with ninhydrin revealed intense spots in the aspartic acid--
glutamic acid region in addition to the tryptophane spot. Electrophoresis
ofglutamic and aspartic acids is nearly as great as the mobility of the compound
responsible for spot V, which may also explain their presence in the hydrolysate.
We have assumed that the second component of the complex - - malonic
acid--is decomposed in the course of hydrolysis. This is also indicated b y
the result of the control experiment, in which a sample of malonic acid was
heated with barium hydroxide under similar conditions. It was not possible
to demonstrate malonic acid chromatographically after this treatment.

Diseussion

Research into the metabolism of indole substances in plants has recently

been advanced b y discoveries of new, relatively low molecular indole com-
plexes. Their physiological significance is not yet clear, it may be expected,
however, that the discovery of these substances will largely contribute to
our knowledge of the metabolism of indole stimulators in plants.
The presence of indoleacetylaspartic acid following the administration of
exogenous indoleacetic acid has been described b y Andreae et al. (GOOD,
ANDREAE and VAN YSSELSTEIN 1956) in a number of plants. According to
our results it is possible to add wheat to these. Indoleacetylaspartic acid appears
in plants only following the exogenous addition of indoleaeetic acid and there
is no report in the literature of its occurrence without external intervention.
Our results are in agreement with this statement.
60 M. KUT.~(~EK, K. ROKOSOVfi,, R. I~ETOVSKY

In addition to indoleacetylaspartic acid and malonyltryptophane discovered

by the Canadian authors, further indole complexes have been described in
the recent literature. They are a labile complex growth substance described
by RAADTS and S6D~Na (1957) in the coleoptiles of oats, and a correlation
inhibitor from the roots of peas studied b y LIBBERT (1955). A natural low
molecular complex, the structure of which is so far best known, is ascorbigen.
According to PROCIt~ZKA, ~ANDAand ~oR~ (1957) ascorbic acid is bound
in it to 1-fl-indolyl-propen-(2)-diol-(2, 3). From the results of one of us (KtT-
TACEK, VALENTA, ICHA 1957 and KUTs et al. 1958) it follows that as-
corbigen is very probably involved in the metabolism of indole growth sub-
stances. The presence of ascorbigen in plants other than members of the genera
Brassica and Raphanus has not, however, been demonstrated as yet. The
isolated occurrence of a spot of a substance similar in chromatographic pro-
perties to ascorbigen in one of our experiments with exogenous tryptophane
was, therefore, all the more interesting. For as yet unexplained reasons it
has not been possible to repeat this finding. It is possible that it depends on
summer conditions. The positive result was obtained in August 1956.
Conjugated derivatives of indole compounds have also been demonstrated
in pathological human urine (also indoleacetylglutamine) (JEPSO~ 1956).
Our work is methodically related to research on ascorbigen. For identi-
fication of the metabolites of exogenous indoleacetic acid and tryptophane
we used some less usual chromatographic systems, both neutral and acid.
Neutral and acid solvents were found to be indispensable for research on
ascorbigen, which decomposes in the ammoniacal systems usually used for
the separation of indoles (KuT~EK, ~[CHA, VALENTA,TuP~ and HOLKOV~i
1958).
Extracts were prepared simply, to prevent b y rapidity in procedure the
formation of artefacts. The preparation of these simple extracts is, however,
inadequate for the demonstration of some substances which are present in
very small quantities.
We turned our attention to spots of indole derivatives which appeared
on chromatograms in system S 1 close to the start. It was shown that in the
case of exogenous indoleacetic acid the spot is caused b y indoleacetylaspartic
acid, both components of which--indoleacetic acid and aspartic acid--were
obtained after hydrolysis. According to a personal communication from
ANDREAE and GOOD (cf. DANNENBURG and LIVEtCMAN 1957), a complex
compound--malonyltryptophane--occurs with exogenous tryptophane. Ana-
lysis of hydrolysates of this spot on chromatograms in system $1 showed
a bound form of tryptophane. The second component of the complex--malonic
acid--could not be demonstrated in view of its lability to hydrolysis.
The results obtained by the method described here, which is different
from that used by the Canadian authors, are in full agreement with the results
of ANDREAE et al. ( A N D R E A E and GOOD 1955, GOOD, ANDREAE and VAN
YSSELSTEIN 1956) and supplement their observations on other biological
material.
We wish to express our t h a n k s to Dr. W. A. Andreae, Science Service Laboratory, University,
Sub Post-Office, London--Ontario, for providing us with samples of indoleacetylaspartie acid
and for information which he kindly gave us during the course of this work.
 M E T A B O L I S M OF E X O G E N O U S TRYPTOPHANE ACID 61

Referenees

ABRAMS, G. J. v o n : A u x i n r e l a t i o n s of a d w a r f pea. - - P l a n t P h y s i o l . 28 : 4 4 3 - - 4 5 6 , 1953.

ANDREAE, W. A., GOOD, N. E.: The f o r m a t i o n of i n d o l e a c e t y l a s p a r t l c a c i d in p e a seedlings.
P l a n t P h y s i o l . 30 : 3 8 0 - - 3 8 2 , 1955.
ANDREAE, W. A., VAN YSSELSTEI~, M. W. H.: S t u d i e s on 3 - i n d o l e a c e t i e a c i d m e t a b o l i s m I I I .
The u p t a k e of 3 - i n d o l e a c e t i c a c i d b y p e a e p i c o t y l s a n d i t s c o n v e r s i o n to 3 - m d o l e a c e t y l a s p a r t i e
acid. - - P l a n t P h y s i o l . 31 : 2 3 5 - - 2 4 0 . 1956.
DANNENBURG, W. N., LIVERMAN, J. L.: C o n v e r s i o n of t r y p i o p h a n - 2 - C 14 to i n d o l e a c e t l e a c i d b y
w a t e r - m e l o n t i s s u e slices. P l a n t P h y s i o l . 32 : 2 6 3 - - 2 6 8 , 1957.
GOOD, N. E., ANDREAE, ~r A., VAN YSSELSTEIN, M. W. H.: S t u d i e s on 3 - i n d o l e a c e t i c a c i d m e t a -
b o l i s m I I . Some p r o d u c t s of t h e m e t a b o l i s m of e x o g e n o u s m d o l e a c e t i c a c i d i n p l a n t tissues. - -
P l a n t P h y s i o l . 31 : 2 3 1 - - 2 3 5 , 1956.
GORDON, S. A., NIEVA, S. F.: The b i o s y n t h e s i s of a u x i n in t he v e g e t a t i v e p i n e a p p l e . I. N a t u r e
of t h e a c t i v e a u x i n . - - Arch. B i o c h e m . 20 : 3 5 6 - - 3 6 6 , 1949. [I. The p r e c u r s o r s of i n d o l e a c e t i c
acid. - - Arch. B i o c h e m . 20 : 3 6 7 - - 3 8 5 , 1949.
GORDON, S. A.: Occurrence, f o r m a t i o n a n d i n a c t i v a t i o n of a u x i n s . A nn. R e v . P l a n t P h y s i o l .
5 : 3 41--378, 1954.
HEMBERG, W.: S t u d i e s on t h e b a l a n c e b e t w e e n free a n d b o u n d a u x i n in g e r m i n a t i n g maize. - -
P h y s i o l . P l a n t . 8 : 4 1 8 - - 4 3 2 , 1955.
JEPSON, J. B.: I n d o l y l a c e t y l - g l u t a m i n e a n d o t h e r in dol e m e t a b o l i t e s i n H a r t n u p disease. - -
B i o c h e m . J. 64 : 14 P, 1956.
KUTJ~EK, M., VALENTA,M., ICHA, F.: U n t e r s u c h u n g e n u b e r d e n A s c o r b i g e n g e h a l t v o n K o h l r a b i
(Brassica oleracea v. gongy'odes) w ~ h r e n d der V e g e t a t i o n u n d d e n Z u s a m m e n h a n g z w i s c h e n
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KUT.~(~EK, M., ICHA, F., VALE~N-TA, M., TuP'#, J., HOLKOVs L.: S t u d i e indolov3~eh derivAtfi
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P h y s i o l . 2 : 169--198, 1951.
LIBBERT, E.: N a c h w e i s u n d c h e m i s c h e T r e n n u n g des K o r r e l a t i o n s h e m m s t o f f e s u n d seiner H e m m -
s t o f f v o r s t u f e . - - P l a n t a 45 : 4 0 5 - - 4 2 5 , 1955.
LIBBERT, E.: Die H y d r o l y s e des , , K o r r e l a t i o n s h e m m s t o f f e s " zu A u x i n . - - P l a n t a ~6 : 256 271,
1955.
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62 M. K U T A ~ E K , K. ROKOSOVA, R. I~ETOVSK~

WILDMAN, S. G., M u ~ , R. M.: Observations on the mechanism of auxin formation in p l a n t

tissues. - - P l a n t Physiol. 24 : 84--92, 1949.
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auxin content of spinach cytoplasmic proteins. - - Arch. Biochem. 14 : 381--413, 1947.

Addres8: Dr. Milan Kuts V:{zkumn~ 6stav rostlinn6 v:~roby, Praha-Ruzyn6. Kv6t~
RokosovA and Doc. Dr. Rudolf l~etovsk:~, Institute of Biology, Czechoslovak
Academy of Sciences, Na cvi6iw 2, Praha-Dejvice.

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