ISOLATION AND PARTIAL CHARACTERIZATION,
or BOUND rams op
mama‘s-«me: ACID IN ZEA MAYS ‘

T119353 for the Degree cf Ph. D.
MICHIGAN STATE UNIVERSITY

Cesar Labarca
19.65

 

LIB? 1R Y
lUlkflii,, IStatc
University

> ErHESIs

  
 
   

This is to certify that the

thesis entitled

ISOLATION AND PARTIAL CHARACTERIZATION
OF BOUND FORMS OF
INDOLE-3-ACETIC ACID IN ZEA HAYS

presented by

Cesar Labarca

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Plant: Physiology

8 In 3 Rm L

Major professor

Date April 26J 1965

0-169

ABSTRACT

ISOLATION AND PARTIAL CHARACTERIZATION
OF BOUND FORMS OF
INDOLE—3—ACETIC ACID IN ZEA MAYS
by Cesagllabarca

Bound forms of indole-3—acetic acid (1AA) have been isolated from
mature sweet corn kernels, and partially characterized. Aqueous acetone
was used in the extraction procedure, following the method described
by Berger and Avery (22). A water-insoluble and a water—soluble frac—
tion, containing IAA in bound form, were obtained.

The water insoluble fraction was a zein-like compound, soluble in
aqueous acetone and aqueous ethanol, with a high molecular weight, and
similar in its properties to the fraction isolated by Berger and Avery.
This fraction has 0.5 to 0.8 % IAA, which is released upon milk alkaline
hydrolysis. Acid hydrolysis of this fraction yielded amino acids and,
probably, reducing monosaccharides. This, and the content of nitrogen,
of about 8 per cent, suggests that this compound could be a glyco-
protein.

The water soluble fraction was further purified by chromatography
on Sephadex 6-25, and its components separated by paper chromatography.
Four water soluble IAA complexes were obtained. Alkaline, acid, and
enzymatic hydrolysis were used to study the composition of these com-
plexes. In all of them IAA is conjugated with inositol, and is released
by mild alkaline hydrolysis. The four complexes were numbered according
to their decreasing mobility in paper chromatography in a n-butanol-
water system. B has 34.0 Z IAA, and contains also inositol, and pro-

1

bably glucose; B2 has 26.2 Z IAA, inositol, and glucose; B3 has 20.5 2

Cesar Labarca

IAA, inositol, glucose, and arabinose; B has 22.2 % IAA, inositol,

4
glucose, and arabinose. The molar ratios of the components of each
complex have not yet been determined.

No estimation has been made of the efficiency of the extraction
procedure, and thus the present estimates of the total content of
bound IAA may be low. There is a minimum of 45 mg of IAA in bound
form per kilogram of mature sweet corn kernels. This amount is equal
to about 10 micrograms of IAA in bound form per kernel, which is about
100,000 times more than the IAA which has been calculated to be present
in one Aygna coleoptile tip.

The conjugation of IAA with inositol, which is known to be a growth

factor and to participate in cell wall metabolism is a new finding and

could have physiological importance.

ISOLATION AND PARTIAL CHARACTERIZATION
OF BOUND FORMS OF

INDOLE-B-ACETIC ACID IN ZEA HAYS

by

(,(Ib' 'le
CesarALabarca

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Botany and Plant Pathology

1965

ii
ACKNOWLEDGMENTS

The author wishes to acknowledge his great indebtedness to Dr.
Robert S. Bandurski for guidance and encouragement. I am also grateful
for the help and critical reading of the thesis by the other members of
my Guidance Committee, Drs. John E. Cantlon, Leo Mericle, and James
Fairley.

I would also like to express my appreciation to Dr. Norman E. Good
for his generous help and suggestions; to Drs. Alex Kivilaan and Lloyd
G. Wilson for their help and encouragement.

I would also like to thank Dr. Peter Nicholls who first suggested.
inositol as a component of the inddeacetic acid complexes.

This work was done while the author was holding a fellowship from
the International Atomic Energy Agency and the University of Chile to
whom he wants to express his appreciation.

The support of the National Science Foundation, under research

grant G—22069, is gratefully acknowledged.

TABLE OF CONTENTS

ACKNOWLEDGMENTS.......................
LIST OF TABLES.............

LIST OF FIGURES..................

INTRODUCTION................................................... 1
A. General considerations of bound forms of IAA............ 1
B. Ascorbigen and glucobrassicin........................... 3
C. Indoleacetyl peptides and glycosides ......... ... ..... ... 5

l. Indoleacetylaspartic acid.. ...... ................... 5
2. Other auxin peptides................................ 7
3. Indoleacetylglucoside............................... 9
4. Indoleacetylarabinose......... ...... ....... ........ . ll
5. Physiological significance.......................... 11
D. Macromolecular complexes ...... .......................... l3
1. Protein conjugates.... ....... ........... ...... ...... 13
2. Protein-tryptophan as a possible source of IAA ...... 16
3. RNA conjugates...................................... 17
E. Other unidentified naturally occurring bound forms...... 18
1. In.Axgna coleoptiles................................ 18

2. Water soluble forms of IAA complexes................ 19

3. Other bound forms of IAA............................ 21

F. IAA complexes in corn................................... 23
EXPERIMENTAL................................................... 28
A. Materials and analytical methods........................ 28

B. Extraction procedure.................................... 29

C. Purification of fraction A.............................. 30

iii

iv

D. Purification and partial characterization of fraction B.

1. Purification of the crude fraction............

2. Separation of fraction B into its components........

3. Partial characterization of the components of B.....

DISCUSSION.............

CONCLUSIONSIOOOO...OOOOIOOOOOO

LITERATURE CITED........

33

33

35

38

58

71

72

LIST OF TABLES

Table l. Fractionation and purification of the water soluble

IAA complexes from 15 kg of sweet corn kernels....... 38

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.
Figure 10.

Figure 11.

LIST OF FIGURES

Schematic outline of the extraction procedure

of IAA complexes from Zea mays......... ..... ........
Diagram of the chromatograms of H2804
hydrolysates of fraction A ................... . ......

Ultraviolet spectra of the four components of
the water soluble fraction B......... ..... ... .......

Diagram of a chromatogram of B B B and B

l’ 2’ 3’ 4
hydrolyzed With NH4OH ..... O O O O O 0 C O I O O O C O C O O O O O O O O O C 0
Diagram of electrOphoretograms of B1’ B2, B3, and B4

in: a) borate buffer pH 9.4; b) glycine buffer pH 9.4

Diagram of a chromatogram of X X2,X3, and X ......

1’ 4

and B

Diagram of a chromatogram of B1’ B2, B3, 4

hydrolyzed with 2.5 N stqgin an autoclave... ..... ..

Diagram of a chromatogram of B1, B2, B3, and B

4
hydrolyzed with snail gut juice................ .....
Diagram of chromatograms of X1 and inositol.... .....
Diagram of an electrophoretogram of X1 and inositol.

Tentative composition of the water soluble complexes

vi

29

32

39

42

48

50

53

55

INTRODUCTION

 

A. General considerations of bound forms of indoleacetic acid.

Although it is now evident that indoleacetyl compounds occupy an
important place in the hormone physiology of plants, it is not yet
known what their chemical form in vivo may be. It was first observed
by Kogl et a1. (79) that alkaline hydrolysis released auxin from seeds.
Indoleacetic acid (IAA) has been isolated from corn meal and the endo-
sperm of immature and mature corn kernels after alkaline hydrolysis (56,
22).

Recently the application of paper chromatographic techniques to
the purification of plant extracts has made possible the detection of
minute traces of active growth-regulating substances, present in too
small an amount to be detected by the older classical chemical methods.
By use of these techniques, IAA has been detected in a wide range of
plant tissues. In some instances IAA was detected on the paper by both
color reactions and biological activity, but many references report
only biological activity in the zone where IAA was expected to occur.
There are also available some reports (18) in which workers were unable
to detect IAA in plant extracts, although it was carefully looked for,
and often a great deal of material was used.

The evidence now suggests that although free IAA probably occurs
in many plant storage tissues, its presence in growing plants can not
be regarded as definitely and universally established. It has been
evident for many years that auxins in plant tissues do not exist only
as free molecules of IAA (18), and there is a growing body of evidence

that it may be Operative as a complex, that is, in a bound form.

Free IAA may be considered, operationally, as that which is readi-
ly and quickly extracted from the plant by methods which are designed
to prevent its release from bound forms. These methods should involve
rapid extraction with solvents that will inactivate enzymes.

Bound forms are regarded as consisting of a molecule of IAA linked
in some way to another molecule. Only recently it has been realized how
easily some of the bound forms or complexes yield free IAA, and thus
how difficult any investigation of the physiological importance of the
free and bound forms must be. This has led to a great deal of confusion
in the interpretation of results. Some of the complexes appear to be so
unstable that it is difficult to prevent the production of free IAA, for
example by hydrolysis, during the processes of extraction and chromato—
graphy. Undoubtedly in most cases the free IAA reported in plant ex-
tracts is really an artifact due to its liberation from some bound form
during the manipulations of extraction and purification.

It has been known for many years that even mildly alkaline condi—
tions liberate IAA from some bound forms in plant tissues. More
recently (163) it has been shown that solvents containing ammonia, which
have been the most commonly used in chromatography of indoleacetyl com—
pounds (133), lead to the breakdown of some of the bound forms of IAA
and to the formation of free IAA and other artifacts. Unfortunately
there are some workers who still rely, almost exclusively, on these
methods in their extraction and purification procedures (72).

Various theories concerning the physiological significance of the
bound forms of IAA have been proposed. Probably the most important
problem is to know whether they are simply immobilized forms of IAA or

are actively concerned in the growth process. Some of these bound forms

have been regarded as precursors of IAA (22), storage forms, detoxi-
fication products (2, 3, 165), and also as being without physiological
importance (160).

From a consideration of both the chemical and physiological evi-
dence on naturally occurring IAA complexes, Bentley (21) concludes that
free IAA is not the auxin physiologically active in normal growth. It
probably occurs free in many tissues, but only in very small amounts
compared with non-ether soluble forms which are held to be the physi-
ologically active forms. The evidence now is fairly strong that IAA
and possibly also other indoleacetyl compounds are produced by break-
down of complex forms of some kind. Some of these naturally occurring
complexes are present in relatively large amounts, especially in seeds,
and only traces of some others have been found. Some of them have been
detected after incubating plant tissues with rather large amounts of
IAA. A few of these complexes have been identified and their structure
is known, but there are still a large number of reports in the litera-
ture about compounds whose composition and physiological importance

have not been established.

B. Ascorbigen and glucobrassicin.

The extraction of ascorbigen from Brassica was reported in 1939 by
Pal and Guha (110) and Sen—Gupta and Guha (121). The compound liberates
ascorbic acid upon acid hydrolysis, while on alkaline hydrolysis it
yields IAA, indole—3-carboxylic acid, and other unidentified indoles
(20). It is itself inactive in the Avena test. Bose and Guha (25) re—
ported the presence of ascorbigen in eight different species of Brassica.
Apparently it has been located only in the Cruciferae and is the only

naturally occurring bound form of ascorbic acid so far isolated (20).

Bose and Guha (25) found that roots and stalks are usually poor in
ascorbigen and ascorbic acid, while leaves and reproductive organs are
especially rich. Kutacek et a1. (84) studied the relationship between
ascorbigen and growth in plants of the Cruciferae family and were able
to demonstrate a very high ascorbigen content in the growing apex, and
also in the young leaves, with a decrease with age in both tissues.
This distribution is similar to that of native auxin in plant tissues.
On chromatography in ammoniacal solvents, ascorbigen breaks down to a
number of indole compounds, several of which have growth regulatory
activity (20). These artifacts would explain the activity found in
extracts of Brassica in chromatograms develOped under ammoniacal condi-
tions.

Kutacek (83) has recently reported that one of the principal in-
dole compounds present in Brassica oleracea is glucobrassicin, and not
ascorbigen as was reported before (84). Other indole derivatives, like
ascorbigen and indoleacetonitrile are probably present, but in very
small amount and he considers that most of them are degradation products
of glucobrassicin. Gmelin (44) has also reported that glucobrassicin
and neoglucobrassicin are the key substances for most of the hitherto
described indole derivatives occurring in cabbage. The sensitivity of
these compounds toward heat or hydrolytic conditions at acidic or alka-
line pH, and their spontaneous cleavage by myrosinase, an enzyme pre-
sent in the press juice of cabbage, makes it understandable that most
of the earlier methods used for extracting and assaying this plant
material, led to complex mixtures of artifacts. Enzymatic degradation
at pH 7.0 produces glucose, sulfate, and probably indole—B-methyliso—

thiocyanate, which decomposes immediately into thiocyanate and

3-hydroxymethylindole. The products of the enzymatic reaction at pH

4.0 are glucose, sulfate, sulfur, H S, and indole—3-acetonitrile.

2
Degradation by chemical hydrolysis follows two pathways, the first
yielding essentially the same products as the enzymatic degradation at
pH 7.0, and the second leading to indole-3-acetonitrile as the first
reaction product; indole-B—acetamide and indole-3-acetic acid appear
gradually on prolonged hydrolysis. Depending upon whether conditions
are alkaline or acidic, a number of byproducts are formed during hy—
drolysis, such as indole, skatole, indole—3—carboxy1ic acid, etc. The
conditions of the process determine which of the degradation products

mentioned above is formed preferentially in enzymatic or chemical de-

gradation.

C. Indoleacetyl peptides and glycosides.

 

(l) Indoleacetylaspartic acid.

 

A study of the metabolism of IAA was first undertaken by Andreae
and Good in 1955 (2, 5, 47, 48), and led to the isolation of indole-
acetylaspartic acid. They showed that when a wide range of plant tissues
were incubated with IAA, the presence of indole-3-acetylaspartic acid
could be demonstrated. The presence of indoleacetylaspartic acid in
tissues of IAA treated plants is well established now and its natural
occurrence has been reported by Klambt (77) in the extracts of tissues
from a number of flowering plants, and by Row et al. (118) in tomato
seedlings. Indoleacetylaspartic acid has been confirmed as the main
metabolite of exogenous IAA in pea tissues by Zenk (164) and other
authors. The evidence shows that the peptide linkage is between the

carboxyl group of IAA and the amino group of aspartic acid.

Wightman (147) found indoleacetylaspartic acid in pea, tomato,
and bean tissue, but not in wheat coleoptile, after incubation with IAA,
indoleacetamide, and methylindole-3-acetate. Winter and Thimann (155)
allowed radioactive IAA to be transported through pea stem sections
and found that part of the activity remained in the sections after two
hours as a bound form which was identified as indoleacetylaspartic
acid. In similar experiments in Aygna coleoptiles no bound forms were
found. Fang et a1. (34) found indoleacetylaSpartic acid in pea shoots
after treatment with radioactive IAA, but only a variety of compounds,
unidentified after chromatography in ammoniacal solvents, were found in
corn tissues. Bennet-Clark and Wheeler (17) found two major products
of metabolism of applied IAA in potato tuber disks, and one of them was
identified as indoleacetylaspartic acid. They suggest the formation of
this compound in the tissue from bound IAA rather than free IAA.
Thurman and Street (142) reported the formation of indoleacetylaspartic
acid after incubation of excised tomato roots with IAA. The complex
was metabolized when the roots were transferred to an IAA free medium.

Andreae, Good at al. (5, 48) have shown that indoleacetylaspartic
acid predominates in the legumes and onion, but not in the grasses,
where indoleacetamide, which later proved to be an artifact, was found.
They found however, that indoleacetylaspartic acid is highly active in
the oat coleOptile test, about equivalent to IAA, but is practically
inactive in peas, where only 1/1000 of the activity of IAA was found
when assayed in the slit pea curvature test or in pea stem elongation
(2). These authors found that both indoleacetylaspartic acid formation
and growth in pea epicotyls became maximal at the same concentration of

applied IAA of about 0.6 x 10—4 M. With lower concentrations almost

all the IAA found in the tissues was present as indoleacetylaspartic
acid, and only with the higher, growth inhibitory concentrations, did
free IAA accumulate in the tissues to any extent. Following the meta-
bolism of two micromoles of radioactive IAA in 20 ml of solution,
Andreae et a1. (4) found that 17 Z of it was conjugated with aspartic
acid after 24 hours. They also reported (6) that the tips of intact
roots accumulated indoleacetylaspartic acid at a rate considerably
greater than epicotyl sections, but excised roots almost entirely lost

this ability unless supplied with sucrose and calcium.

(2) Other auxin peptides.

 

Incubation of pea epicotyls with radioactive naphthaleneacetic
acid (NAA) for 24 hours, resulted in the conjugation of up to 95 Z of
the NAA taken up as naphthylacetylaspartic acid. The synthesis of the
peptide proceeded via the free L—aspartic acid (164). The authors
considered that the uptake of NAA in epicotyls represents an active
process; the concentration of the acid in the tissue exceeding twice
the concentration in the medium. The formation of naphthylacetyl—
aspartic acid was found to be an adaptive process which could be induced
by preincubation with NAA, IAA, 2,4-D, and benzoic acid. An adaptive
(L)-aspartic-N-acylase had been postulated by Zenk (164).

Sfidi (135) in a study of the induction of the formation of com—
plexes between aspartic acid and IAA and NAA, reported an effect of
pretreatment with IAA and six other compounds on the subsequent forma-
tion of indoleacetylaspartate and naphthylacetylaspartate. He found
that IAA, NAA, 2,4-dichlor0phenoxyacetic acid, 2,3,6—trichlorobenzoic
acid, and S-carboxymethyl—N,N-dimethyldithiocarbamate, which are active

as growth regulators, are all highly active as inducers. By contrast,

2,4-dichlor0phenoxyisobutyric acid, and 3,5-dichlor0phenoxyacetic acid,
which possess little or no activity as growth regulators, were inactive.

The presence of malonyltryptophan has also been reported in plant
tissues. It was first found by Good and Andreae (47) in Spinach leaves
incubated in a tryptOphan solution, and later, as a naturally occurring
compound in tomato, spinach, pea, and oats. The presence of D—trypto-
phan in this conjugated form has been reported (117,166, 77), and it
has been shown to be present in the vegetative tissues and fruits of
plants of 14 different genera (166). In fruits it is found that
intoleacetylaspartic acid and malonyltryptoPhan are produced especially
during maturation (165). This was first observed in apple fruits (117).

Thurman and Street (139) have reported the possible presence of
indoleacetylglutamic acid in excised tomato roots exposed to IAA.

Andreae and Good (3) in a study concerning the metabolism of applied
carboxylic acids, found that when pea epicotyl sections were incubated
in solutions of indoleformic (indole-3—carboxylic) acid, indolepropionic
acid, indolebutyric acid, benzoic acid, and 2,4—dichlorophenoxyacetic
acid, all the corresponding aspartic conjugates, except indoleformyl-
aspartic acid, were found in the tissues.

Zenk (164) has presented evidence suggesting that these metabolites
are enzymatically formed from carboxyl activated auxin and L—aspartic
acid. He also reported (162) on an ATP dependent formation of IAA-
coenzyme A esters, catalyzed by octanoate—thiokinase, an enzyme from
liver mitochondria. Indoleacetyl-AMP was an intermediate in the forma—
tion of indoleacetyl-GOA, and this was shown to be an intermediate pro-
duct in the enzymatic formation of the indoleacetylglycine. The

esterification of IAA with CoA, as a mechanism of action of the plant

hormone, has been previously postulated by LeOpold (88).

Winter and Street (154) found a 5~hydroxytrypt0phan peptide, con-
taining glutamic acid~arginine~alanine-valine-leucine, and 5-hydroxy—
tryptophan, in "staled" tomato root culture medium. They isolated 2.2
mg of the peptide from 20 liters of culture medium after exhaustive
extraction with ethylacetate. This peptide is highly active in pro—
moting both the extension growth of coleoptile segments and the ex-
pansion of root cells. Street at al. (134) have reported the presence

of indole compounds in extracts of seedling roots of Vicia faba and

 

Phaseolus multiflorus which resemble the 5—hydroxytryptophan peptide
in their Rf values in paper chromatograms, their movement in paper
electrophoresis and their biological activity. They postulate that
such compounds could be closer to the active growth regulating mole-

cules than the free indole-containing compounds, and could arise

directly from biologically active indole-containing proteins.

(3) Indoleacetylglucoside.

 

Zenk (163), in 1961, isolated and identified a conjugated form of

IAA and glucose from leaves of Colchicum neapolitanum incubated in an

 

IAA solution. The structure of the glucoside was l-(indole-3—acetyl)-
B-D—glucose. This conjugated form was also isolated by Klambt (78)
from wheat incubated with IAA and, together with the formation of
indoleacetylaspartate, is probably a general mechanism of conjugation
of exogenous IAA in plant tissues.

Zenk (165) has shown that indoleacetylglucose can be synthesized
from IAA and uridinediphosphoglucose by a soluble enzyme system from

lentiles similar to the one used by Jacobelli et a1. (73).

10

The glucose—IAA bond, being a hemiacetal—carboxylic anhydride, is
more reactive than an ester bond, and can donate the acyl group to
ammonia, amines, hydroxylamine and alcohol (165). At pH 7.0 the re-
action with ammonia and hydroxylamine leads to the formation of indole-
acetamide and indoleacetylhydroxamate. Incubation of the glucoside
with amino acids or ethanol at alkaline pH yields, in addition to IAA,
the corresponding acetylated amino acids or the indoleacetic—ethyl
ester. Reports in the literature of the occurrence of indoleacetamide
are considered erroneous and probably due to artifacts of chromatography
and isolation (165).

Andreae and Good (3) in a study of the metabolism of applied
carboxylic acids, found that when pea epicotyl sections were incubated
in solutions of indoleformic (indole-3-carboxy1ic) acid, indolebutyric
acid, benzoic acid, or 2,4—dichlorophenoxyacetic acid, all the corres—
ponding amides, except 2,4-dichlorophenoxyacetamide were present in the
tissues. As was later shown by Zenk (163) the use of ammonia in the
development of the chromatograms probably led to the formation of the
amides from the glucose esters which almost certainly were the compounds
actually present in the tissues.

A conjugation product of NAA, l-(d-naphthylacetyl)-B—D-glucose,
was isolated and identified by Zenk (164) from pea epicotyls incubated
with radioactive NAA, and its structure was verified by synthesis. In a
similar experiment Klambt (78) found the glucose ester of benzoic acid in
wheat.

In in vitro studies with polyphenol oxidase, LeOpold (58) separated
three pigments which seem to be addition products of IAA and quinones of

catechol, chlorogenic acid, and caffeic acid.

ll

(4) Indoleacetylarabinoside.

 

Analysis of several Ehrlich positive spots on paper chromatograms
of corn fractions with growth promoting activities, led to the isolation
by Steward et al. (123, 131) of a non-crystalline but chromatographically
homogeneous substance. Its probably structure, based upon reactivity,
hydrolysis products, and other properties, is l-(L-arabinosyl)-indole-
3—acetate. This compound was found to promote a somewhat greater degree
of cell division in carrot tissue than did IAA, whereas in the promotion
of elongation in the Avena mesocotyl it has only about 1 Z of the

activity of IAA.

(5) Physiological significance,

 

There has been some question as to whether the compounds formed
after feeding IAA, or other synthetic auxins to plant tissues actually
represent naturally occurring compounds. As Bentley (21) points out, it
is not clear whether the application of large amounts of auxin to a
tissue leads to reactions that would not normally occur with the much
lower levels of endogenous auxins. In this respect, Mfiller (101) dis-
tinguishes between two groups of compounds: a) Derivatives which appear
in the tissues after incubation in solutions of IAA and other indole
compounds, and are broken down again by true enzymatic degradation.
These can be considered as native in the tissues. b) Compounds which
appear, probably because of non-physiological conditions, are not broken
down again and accumulate in the tissues. In this second group he
classifies the indoleacetylaspartic acid, the indoleacetamide, and other
unidentified substances.

Andreae, Good, et a1. (2, 3), and Zenk et al. (165) agree in

12

considering that the conjugated forms of IAA with glucose and aspartic
acid found in their experiments are the products of detoxification
processes which dispose of an excess of IAA. In this respect the
mechanisms could be compared with the formation of indoleacetyl-L—
glutamine and indoleacetylglucosiduronic acid, which appear to be
detoxification products of IAA in humans (74, 75, 127). A large amount
of internal indoleacetylaspartic acid apparently does not interfere
with the elongation of roots (1).

Zenk (165) has studied the distribution of the principal con-
jugation systems in about 170 species by applying rather high IAA
dosages. The two principal pathways found were conjugation with aspartic
acid and with glucose. Conjugation with glucose seems to be the more

primitive way, being present in Bacteria, Fungi, BryOphyta, and

 

Sphenopsida. Lycopsida, and Pteropsida, including Filicinae, Gymno—

 

 

 

spermae and Angiospermae, still have the ability to conjugate IAA with

 

glucose, but they also have a second system, the conjugation of IAA

with aspartic acid. Among the Angiospermae, the Liliales seem to favor

 

most the pathway of glucose conjugation. Most species of plants made
use of both types of conjugation, but since there exist species which
apparently form only indoleacetylaspartic acid or only indoleacetyl-
glucose, it is unlikely that either conjugate is an essential part of
the growth induction mechanism. Zenk therefore assumes that both con-
jugates are true detoxification products.

There is only qualitative data to date about malonyltryptOphan,
but since it has been identified as the detoxification product of
D-tryptophan (166), Zenk has suggested the following hypothesis (165):

At the point where growth stops in a fruit and IAA is no longer consumed,

13

it is converted to indoleacetylaspartic acid or indoleacetylglucose.
Further, in order to block IAA production, some indole precursor of IAA
is transformed to D-tryptophan, which is immobilized and detoxified as
malonyl-D-tryptophan. This would lead to the accumulation of the malonyl
derivative in mature fruits. Thus, according to Zenk, both the produc-
tion and the action of IAA may be stopped in the fully developed organ.
Based on the information available about the induction of formation
of indoleacetylaspartate after the application of IAA or other growth
regulators to plant tissues (165), Zenk concludes that the formation of
indoleacetylglucose in plants seems to be a mechanism for very rapid
detoxification of aromatic carboxylic acids during a time when the
conjugation system utilizing aspartic acid is being adapted in the
plant. The glucose ester of IAA is not stable in the cell after the
aspartic acid conjugating system has been adapted, but becomes eventually

transformed into indoleacetylaspartic acid.

D. Macromolecular complexes.
(1) Protein conjugates.

It has been suggested in the literature that auxins exert their
effect on cell elongation by combining with a protein, thus yielding an
active enzyme complex (60, 148). Presumably the level of this enzyme
than controls some aspect of growth. Consequently there has been a
great deal of interest in IAA—protein complexes.

The existence of auxin-protein complexes was first reported by
Wildman et al. (153). On the basis of extensive investigations on
Spinach leaf proteins, they arrived at the conclusion that IAA combines
with one of the major leaf proteins, a fraction which has phosphatase

activity. The IAA- protein presumably played a role in the transfer of

14

energy required for growth. IAA was not dissociable from the protein by
repeated fractionation with ammonium sulfate and remained in the molecule
after removal of the phosphorus containing substances (150). Yields of
auxin on treatment with alkali or proteolytic enzymes (153, 53) approach
the ratio of one mole of IAA per mole of protein of 200,000 molecular
weight. Schoken (120) obtained optimum yields from this fraction by
refluxing at 1000C in 0.1 N NaOH.

Gordon (49) found auxin associated with each of the five major
proteins isolated from the wheat grain. Two types were obtained, acid
labile-alkali stable, and acid stable-alkali labile. Only the first of
them, or IAA type, was obtained upon alkaline hydrolysis. Avery et a1.
(11) obtained auxin yields of the same magnitude from wheat with high
and low protein content. They found the highest yields at pH 9.3 to
10.5, and marked destruction occurred by autoclaving with l N NaOH, in
contrast with the alkali stable auxin from corn endosperm. They
postulate the presence of two precursors which are converted into two
auxins upon alkaline hydrolysis and differ markedly in their stability
toward alkali.

In 1944 Berger and Avery (22, 23) reported that 95 Z of the total
auxin activity in sweet corn kernels was attributable to a bound form
of IAA, extractable with aqueous acetone, and which, in its purest form,
had an IAA content of 3.7 Z. This IAA-complex was insoluble in water
and was readily hydrolyzed by mild alkaline treatment to yield IAA. It
did not release IAA on treatment with proteolytic enzymes. The solu-
bility properties, nondializability, and other characteristics of the
purified fraction suggested a macromolecule, probably a protein, but the

nitrogen content was low, about 4.7 Z.

15

Attempts to obtain the conjugation of IAA with protein have been
made in vivo and in vitro by applying rather high concentrations of IAA.
Siegel and Galston (124) have claimed the in vivo binding of IAA in pea
root apices, and an:h1vitro binding in the presence of ATP to a protein
fraction of pea brei. The protein complex formed was stable to boiling,
acid and alkali, and was not destroyed by IAA oxidase. The compound was
rapidly formed during the initial hours of incubation and precipitated
by trichloroacetic acid. The evidence for this protein conjugate however,
has been questioned by Andreae et a1. (7, 1), who found all the
colorimetrically determinable IAA metabolites in IAA treated pea
homogenates and tissues to be small molecules soluble in 70 Z ethanol,
and could find no evidence of a metabolically produced IAA-protein
complex. They believe that the IAA-protein complex reported by Siegel
and Galston is not a product of plant metabolism, but an artifact of the
trichloroacetic acid precipitation procedure.

Zenk (165) has also reported the formation of an IAA-protein com-
plex in vivo by applying carboxyl-ClA—labeled IAA to pea epicotyls. This
IAA was not removed from the protein complex by Sephadex filtration, but
a quantitative recovery of the radioactivity was obtained upon alkaline
hydrolysis.

Skoog and Thimann (126, 141) observed that protein hydrolyzing
enzymes were capable of producing auxins from various protein materials.
The yield of auxin from Lgmg§_was increased greatly by short incubation
with chymotrypsin or trypsin. Crystalline trypsin brought about only a
small yield, while chymotrypsin was highly effective. No auxin was
liberated from £2233 either by extraction with water or alkaline hydro-

lysis in an autoclave. They concluded that auxin in Lemna is bound to

16

protein or to a protein-like substance. Wildman and Bonner (144) working
with spinach proteins found that trypsin gave almost as high auxin yields
as alkaline hydrolysis when the trypsin treatment was preceded by a

treatment with pepsin. Muir (100) reported production of auxin by using

pancreatin.

(2) Protein-tryptophan as a possible source of IAA.

The possibility that the auxin obtained upon alkaline treatment of
proteins arises from tryptophan has been considered. In theory any
protein containing tryptophan could produce IAA upon hydrolysis and
oxidation. However there is little evidence correlating IAA release

I
with tryptophan content. Kulescha (81) found that gelatin, agar, and
gum arabic, compounds which do not contain tryptophan, yielded con-
siderable quantities of auxin upon treatment with trypsin. Alkali
treatment of tryptophan yields at the most very small amounts of IAA
(9, 120, 30). Although traces of IAA may be produced from tryptophan in
a number of proteins (9, 120), it has been calculated that the tryptophan-
IAA conversion is less than 0.01 Z (9). Treatments with various enzyme
preparations have yielded equally small amounts (52, 149, 152). It is
extremely unlikely that the mild conditions used in most of the alkaline
hydrolysis of the auxin complexes were sufficient to break the peptide
bonds in the protein molecule (23), yet under those conditions highly
active indole auxins are obtained from indole-containing complexes.

Although protein materials may conceivably serve as an IAA source,
auxin production has not been generally associated with protein break-

down in the plant. In fact, auxin production in growing tissue may more

logically be associated with protein synthesis.

17

Some bacteria very readily convert tryptophan to IAA (132).
Acetobacter xylinum cells incubated with DL-tryptOphan produced IAA, and
the yield was of the order of 5 Z (87). IAA was also detected when tryp-
tamine or tryptophan was added to the medium where the fungus Diplodia
natalensis was growing (15). In the higher plants only a minute amount
of active auxin appears to be produced from tryptophan or tryptamine,
according to Stowe (132), much less than the amount found in plant ex-
tracts (160). Wildman et al. (152) infiltrated tryptophan into living
spinach leaves, and found an increase in the ether extractable auxin
after 3.5 hours. They also reported rapid conversion of tryptophan to
auxin by a lyophilized, cell free extract of spinach leaves, and demon-
strated the reaction to be enzymatic. In general though, work on the
in vitro conversion of tryptOphan to auxin has shown a very low yield
(20, 103, 156). Gordon and Paleg (51) found that phenols, under
conditions leading to their oxidation, react with tryptophan to form
IAA. The reaction is catalized by the polyphenolase enzymes of mung
bean, oat, and sunflower, and occurs spontaneously at an alkaline pH.
They considered the reaction as possibly more important in wounded plant
tissue, where lysis and activation of the phenolase system takes place,

than in normal metabolism.

(3) RNA conjugates.

Meudt and Galston (96, 97) have reported the formation of a com-
plex between RNA and IAA in shoots and roots of etiolated pea seedlings.
Incubation of IAA with peroxidase yielded an IAA derivative of an un-
known structure, which forms a complex with pruified RNA from pea shoots
at pH values of 4.8 or below. Apparently the complex was stable at

higher pH, and the IAA derivative was firmly bound to the RNA, when the

18

RNA was precipitated by trichloroacetic acid or by other means. The
complex was not formed when IAA itself was added to RNA, or when RNA
was first treated with RNAase. They also found an RNA fraction re-
sembling the in vitro complex after dextran gel filtration of etiolated
pea epicotyl homogenates previously exposed to substrate levels of IAA.
They interpreted this as evidence of the in vivo occurrence of the

process.

B. Other unidentified naturalLyoccurringibound forms of IAA.

(1) In Avena coleoptiles.

Much interesting work has been carried out on the state of the auxin
in A3333 coleoptiles. There is evidence that the major part of the auxin
probably exists in the form of a complex. Since the Aygna coleoptile is
so widely used as a bioassay for auxins, it is obviously important to
know the nature of the native auxins of the coleOptile.

A group of German workers (42, 98) found that the auxin in Aygga
is released from an antiauxin complex with inhibitory properties. This
inhibitor—auxin complex was demonstrated to be present in seedlings of

Zea mays, Avena, and tubers of Solanum tuberosum.

 

Terpstra (138, 139) has carried out chromatographic examinations
of the auxins extractable from coleOptiles. She suggests that the free
form in the intact tissues may be present in neglibile amounts and that
its exact quantity would be of little physiological interest. She demon—
strated that the auxin in extracts of ground coleoptile tips is present
as a complex which is water soluble but ether insoluble. The complex is
unstable and yields IAA very easily.

Raadts and SBdung (116) carried out an extensive chromatographic

19

study of auxin obtained from coleoptile tips by diffusion. They found
IAA and an inactive, labile substance that appears to be an addition
compound of IAA with something else. It very easily breaks down, and
they suggest that the IAA obtained in their extracts is actually an
artifact arising from breakdown of this compound.

Goldsmith and Thimann (46) found considerable retention of radio-
activity in.Ayg§§_coleoptile sections after removal of the source of
radioactive IAA. They think that the immobilized IAA is probably very
tightly bound.

The intracellular localization of native auxin in the Aggga coleop-
tile has also been studied by Yamaki and Nakamura (159, 103). Both free
and bound auxin were localized in the cytoplasm and identified as indole

compounds. There was about twice as much bound auxin as free auxin.

(2) Water soluble forms of IAA complexes.

Numerous water soluble, but relatively ether insoluble, auxin
complexes, which yield IAA by treatment with acid or alkali, have been
found. (IAA is very ether soluble and partitions almost quantitatively
from water into ether.) These substances often appear in large quantities
relative to the amount of free IAA in the tissue.

Avery et a1. (10) reported that about 90 Z of the total auxin con—
tent of dormant maize endosperm exists as a physiologically inactive com-
pound, readily soluble in water but not in ether, and easily converted
to auxin at alkaline pH. A water soluble precursor which gives IAA by
ether extraction after mild heat treatment has been reported in a number
of plants, especially maize seeds by von Denffer (32). This seems to be
comparable with the IAA precursor reported by Housley and Bentley (71).

Britton and Housley (27) found a bound form of IAA in the aqueous, ether

20

insoluble extract of immature corn kernels which possessed marked growth
promoting activity. This compound was hydrolyzed by acid and alkali and
by chromatography in ammoniacal solvents to give IAA.

Farrar et a1. (35, 36) found only part of the auxin activity in
the ether extract of immature corn kernels. The juice expressed from
the ether extracted kernels was still highly active biologically, and
most of the activity remained in the aqueous phase after exhaustive
ether extraction under acidic conditions. A number of substances were
found which gave a purple color with Salkowski reagent quite distinct
from the red given by IAA. These substances decompose on paper
chromatography in solutions containing ammonia, and one of the break-
down products is IAA. Farrar called them zeanins, and described them
as "plant growth substances occurring in Zea mays giving a purple re-
action with the Salkowski reagent, and breaking down on chromatography
in ammoniacal solutions to give II ". All the substances are more
polar than IAA and run behind it in butanol-water chromatography.
Farrar considers these substances different from native bound auxin and
suggests the possibility that they are intermediates between free and
native bound IAA.

Thimann and Skoog (140) found that water was essential for rapid
and complete extraction of auxin in all the materials studied. From
this they concluded that water was necessary for the hydrolytic
liberation of auxin from its bound forms in the tissues. In Lgmgg
they could obtain repeatable results by successive extractions with
ether, but the process required several months to reach completion.
Similar results have been obtained by other workers (109, 141). Avery

et a1. (14) in a comparative study of methods for single solvent ex?

21

traction of growth hormone from plant tissues, found very wide
differences in yield depending on the solvent used and the extent of
hydration of the tissue. Water gave the highest yields, and water and
alcohol gave the most reproducible results. They proposed successive
extraction with different solvents for assaying total hormone content.
Deverall and Daly (33) found that normal mature wheat leaves
converted approximately 60 Z of exogenous carboxyl-labeled IAA in six
hours into four radioactive water soluble compounds which seem to be
bound forms of IAA and were different from each other.
Britton et al. (28) in a paper chromatographic study of extracts
of tomato roots, found three water soluble, ether insoluble compounds.
Two of them, called X and Y, act as precursors or complexes, yielding
the third, 2, and yet they are themselves biologically active and
mutually interconvertible. They also found two interconvertible spots
on chromatograms of water soluble extracts of immature corn kernels (27).
A similar behavior has been noted with auxin obtained from algal extracts
by Bentley (19), although in these extracts only components X and Z
were present in quantity. Audus and Gunning (8) have also reported

four interconvertible auxins in pea roots.

(3) Other bound forms of IAA.
Tandler (136, 137) has reported the presence in the giant uni-

cellular alga Acetabularia, of intracellular crystals containing the

 

indole ring. Alkaline hydrolysis of ethanol extracted algae liberated
substantial amounts of indole compounds. Insolubility in acid media,
in concentrated ammonia, in organic solvents, and in strong alkali,
after formaldehyde treatment, probably indicates a high molecular

weight compound. The crystalline derivative has also been found in

22

several other genera of the Dasycladaceae. It is localized mainly in
the central vacuole.

Muir (100) has reported that large amounts of plant growth hor-
mones are present in bound form in pollen grains of several species.
It can be converted to an active form by alkaline hydrolysis. In dried
ovary tissue of Nicotiana and Antirrhinus, hormones are present only in
an inactive form which can be activated by incubation at pH 8.5 and in
the case of Nicotiana, by a water extract of Nicotiana pollen in acid
medium in vitro.

Srivastava (128, 129) has reported the extraction of auxin from
corn kernels in the early milky stage with cold ether, or ether at
25°C followed by cold ethanol. On paper chromatographic separation,
he obtained three indole compounds by ether extraction of neutral
aqueous solutions of the corn extracts, six more by ether extraction
of the acid solution, and another six indole compounds remained in the
aqueous phase. The water soluble, ether insoluble compounds, gave IAA
on autoclaving under alkaline conditions, and he suggested the presence
of arabinose in one of them. Based only on biological activity he
estimated the total amount of IAA in this plant material as more than
100 mg of IAA per kilogram of fresh weight. Apparently no further
attempt was made to characterize these fractions, in spite of the very
large amount of them available.

Vlitos et al. (144, 145, 146) could only detect IAA in barley and
tobacco tissue after alkaline hydrolysis, which indicates the existence
of an IAA complex. Kbves and Sirokman (80) found two Ehrlich-positive

radioactive compounds with the R of tryptophan and IAA in chromatograms

f

of extracts of pea seedlings grown for 24 hours with radioactive phosphate.

23

Hamilton (58, 59) found IAA in 80 Z ethanol extracts of corn ker-
nels, as well as in sugar beet roots and leaves. He suggests that IAA
may occur as a labile complex in the ethanol extracts of those tissues.
An 80 Z ethanol-soluble, water insoluble fraction from corn kernels
was found to release IAA upon alkaline hydrolysis. It appears this
fraction may be proteinaceous in nature, and probably similar to the
complex reported by Berger and Avery (22).

Hamilton (59), investigating the presence of free IAA in growing
tissues, extracted 11 kg of 5 day old etiolated corn shoots using the
acid ether extraction procedure, and did not find any traces of IAA,
unless the material was treated with alkali. In his experiments he
added CIA-labeled IAA to permit recovery calculation.

In the studies of Kuraishi and Muir (82), the diffusible auxins
of several plant species were shown to be different from free IAA, but
had the same Rf as IAA on paper chromatograms developed with solvents
containing ammonia; probably some labile complexes were broken down
under these conditions.

Scott and Jacobs (121) reported in a very recent paper that the
only compound which diffused into agar blocks from the apical shoot
tips of Coleus Blumei was IAA. No bound forms or any other compounds
with growth regulating activity were found. However, they extracted
the agar cubes with ether, and furthermore, they used ammoniacal sol-
vents for chromatography. Using such extraction procedures it would be
possible to detect only those bound forms soluble in ether and resistant
to breakdown by ammonia.

F. IAA complexes in corn.

Cereal seeds are a rich source of IAA in bound form. Hatcher (62,

24

63, 64) found that among the species of seed tested, corn had the highest
auxin content, followed by oat, rye, wheat, and barley. Probably the
best known naturally occurring auxin complex is that present in corn
kernels. The nature of this complex was extensively studied by Berger
and Avery (23), who reported a total concentration of IAA equal to
about 140 mg per kilogram of sweet corn kernels. Of this they were
able to isolate about 14 mg of IAA per kilogram.

There are marked fluctuations in the content of extractable auxin
during the development of the corn kernel, and this has been studied
by several authors (13, 50, 62, 63, 64, 91). Preceding pollination,
the content of readily extractable auxin of the ovary is low, but starts
rising immediately after fertilization, rising steadily as the embryo
develops, to reach a maximum in from one to three weeks. Then the con—
tent of extractable auxin falls as the seeds ripen. Other authors (66)
using different methods of extraction, reported complete disappearance
of the auxin in fully ripened kernels. A similar pattern of change in
auxin levels has been noted for many species, both monocotyledons and
dicotyledons (12, 62, 63, 64, 91, 92, 104, 105). This marked decrease
in maturing seeds has been ascribed to the conversion of free IAA, or
an easily extractable water soluble form, to a more tightly bound form
(130) subsequently available to the embryo upon germination (55).

Avery et a1. (11) found that the bulk of the auxin was present as
a "precursor" in corn kernels. Yamaki and Nakamura (160) found only
negligible amounts of bound IAA in the embryo, but an abundance in the
endosperm of corn. Hatcher (63) reports an increase in the amount of
auxin as the grain ripens, and its location in the endosperm and

aleurone layer in rye. Polevoi (113) has shown a bound to free auxin

25

ratio of 120:1 in dry corn kernels, and a similar ratio is reported by
Zinovev (167).

During germination there seems to be a decrease in bound auxin in
the endosperm and an increase of water soluble auxin in the corn embryo
and seedling (14, 55, 65, 113, 167). Apparently the same phenomenon
occurs in barley and rice (90, 125).

The fluctuations of the auxin content in corn kernels suggest that
IAA is present in early stages of the development of the seed in a
water-soluble bound form, easily extracted and hydrolyzed. Probably
this is gradually converted to the high molecular weight, water-insoluble
form, which is present in the mature kernel. Upon germination of the
seed, the high molecular weight bound form may gradually liberate, as
the endosperm tissue is used up, a water soluble form which is then
found in the embryo and the seedling.

The fraction extracted with aqueous acetone by Berger and Avery
(22), as well as the one extracted with aqueous ethanol by Hamilton
(58), had many properties similar to zein, the water insoluble protein
of corn kernels. Undoubtedly the fraction of both Berger and Avery,
and of Hamilton, contained at least a high proportion of zein, since
their methods of extraction were similar to those sometimes followed in
the deliberate extraction of zein (95).

Zein constitutes about one third of the total protein of the mature
corn kernels (40, 99) and is localized almost exclusively in the endo—
Sperm (156, 157, 158) where it accounts for about 40 to 60 Z of the
total nitrogen. There is no agreement about the molecular weight of
this protein (99) but it seems to average about 35 to 40,000. Apparently

there are at least three components in zein with different molecular

26

weights (45). There is no tryptophan in zein, but the contents of
glutamic acid, leucine, proline and alanine are very high. According
to Mosse (99) it could be assigned the approximate general formula of
GluzLeuzProllA1a1X4, where X indicates other aminoacid residues. The
elevated number of its non—polar residues and the exceptional scarcity
of its ionic groups probably accounts for several of its peculiar
properties: insolubility in water; weak hydration; solubility in a wide
range of organic solvents; and resistance to denaturation, even at
temperatures of 100 to 180°C.

The globulins and glutelins of the corn kernel appear to be syn-
thesized at a relatively uniform rate throughout the growth period
(161), but zein is nearly absent in the very immature corn kernel and
is synthesized at a very rapid rate as the corn approaches maturity.
The rapid increase in zein nitrogen to total nitrogen ratio, from about
3 to 40 Z, is almost exactly paralleled by the decrease in water—soluble
non-protein nitrogen. Sweet corn is apparently similar to field corn
in zein content.

There seems to be a parallel between the synthesis of zein in
corn kernels at the expense of the non-protein water-soluble nitrogen
in the late stages of the develOpment of the grain, and the formation
of a water-insoluble bound form of IAA, with the simultaneous dis-
appearance of a water soluble form.

The fraction isolated by Berger and Avery (23) was believed to be
a protein but its 4.7 Z nitrogen content is very low for a protein.
From the studies of Zenk (163) and some less adequate reports (123,
128, 131) we know of the conjugation of IAA with sugars, and therefore

the possibility exists that Berger and Avery were actually dealing with

27

a glycoprotein.

Recently Hochstrasser (67, 68, 69) in studying the glucoproteins
of barley endosperm, has been able to isolate peptidyl—polysaccharides
upon hydrolysis of barley albumin. These peptidyl-polysaccharides were
found to contain aspartic acid as the principal aminoacid, and xylose,
arabinose and glucose. He concludes that these compounds are probably
involved in the bridging link between protein and carbohydrate. The
investigation of similar glucoproteins in the endosperm of corn and
other cereals and its relationship with IAA apparently has not been
undertaken yet and it could prove to be extremely interesting.

Although there is now a good deal of information about the presence
of bound forms of IAA in bound tissues, very little is known about the
chemical structure of the numerous IAA complexes reported or, in many
cases, only suggested. It may be that some, or even most, of the sub-
stances found differ in their prOperties as a result of the isolation
procedures employed. Thus the possibility exists that many of these
complexes are identical. As long as the chemical identity of these
compounds remains unknown, the investigation of their physiological
importance is difficult, and even the isolation procedures will remain
empirical.

Mature corn kernels are one of the richest sources of IAA among
plant materials and the prupose of the work described in the next sec-
tion has been to characterize the bound forms of IAA present in this
tissue. The fraction isolated by Berger and Avery (22) from sweet corn
was selected as the starting material, since it is present in large

amounts and it is readily extracted by a rather simple procedure.

EXPERIMENTAL

 

A. Materials and analytical methods.

Mature kernels of a white sweet corn, Stowell's Evergreen Hybrid,
were supplied by the Lansing Farm Bureau.

A modified Salkowski reagent was employed for the colorimetric
determination of IAA. The Salkowski reagent contained 15 ml of 0.5 M
FeC13, 300 ml of concentrated H2804, and 500 m1 of water. Three milli—
liters of this reagent and two milliliters of an aqueous IAA solution
were mixed, and color intensity measured after 30 minutes using a #54
filter in a Klett-Summerson colorimeter, or in a Beckman spectrophoto-
meter at 530 millimicrons.

The solvent used for chromatography of the IAA complexes was
n-butanol:acetic acid:water, 5:1:2.2 (163). This solvent, and ethyl
acetate:pyridine:water, 8:2:1, were used in the chromatography of
sugars and oligosaccharides-

The detection of IAA and IAA complexes on paper chromatograms was
made by using an Ehrlich dipping reagent, prepared by dissolving 2 g
of paradimethylaminobenzaldehyde in 100 ml of concentrated HCl:acetone,
10:90. Sugars and oligosaccharides were detected on paper chromatograms
with silver nitrate (143), aniline phthalate and p-anisidine (70, 111).

Glucose was determined by the glucose oxidase method (119), using
a "Glucostat" reagent from Worthington Biochemical Corp.

The beta—glucosidase was from Sigma Chemical Corp., and Nutritional

Biochemical Corp. The Snail Gut Juice (Suc d'Helix Pomatia) was from

 

Industrie Biologique Francaise, S.A., Gennevilliers, France.
Throughout this work purity of compounds is calculated on the basis

of the content of IAA obtained after alkaline hydrolysis.
28

29

B. Extractiongprocedure.
The extraction procedure described by Berger and Avery (22) was

used to obtain the crude fractions.

DRY KERNELS OF SWEET CORN

ground and extracted with
50Z acetone - 3 times

AQUEOUS ACETONE EXTRACT

shaken with NaCl
water phase washed
with acetgne 3 times

'9 1
WATER
PHASE ACETONE PHASE

 

concentrated

at low pressure
L

V
AQUEOUS l
SUPERNATANT PRECIPITATE
FRACTION B FRACTION A

 

Figure 1. Schematic outline of the extraction procedure of
IAA complexes from Zea mays.

The steps followed in the isolation are summarized in Figure 1,
and were as follows: Corn kernels were ground and extracted with 50 Z
aqueous acetone, v/v, for 48 hours, using two liters of solvents per
kilogram of corn, with occasional stirring. The suspension was filtered
with suction to give a clear yellow extract, and the residue reextracted
as above one or two more times. The combined acetone extracts were
saturated with sodium chloride and shaken vigorously. Following separa—
tion of the two solvent layers, the upper (acetone phase) was saved and
the water phase reextracted three times with a volume of fresh acetone
equal to the volume of acetone removed. The combined acetone fractions
were concentrated in vacuo in a flash evaporator to yield an aqueous

supernatant solution and a zein-like precipitate. The precipitate is

30

called fraction A, and the aqueous supernatant fraction B.

C. Purification of fraction A.

The orange, gummy precipitate obtained upon concentration of the
acetone fraction, was redissolved in 50 Z acetone, and reprecipitated
by pouring the solution into about 40 times its volume of cold 2 Z NaCl
solution. The resultant precipitate was collected on a thick filter
cloth, the water squeezed out, and the precipitate dried in a desiccator
over P205. The dry sample was ground in a mortar to a fine yellow pow-
der.

Fraction A is insoluble in water, ether, acetone, and alcohol, but
is soluble in aqueous acetone and aqueous alcohol. When this fraction
was suspended in water, KOH added to pH 11.5 to 12.0, a clear yellow
solution was formed, with simultaneous release of free IAA. After
adjusting the pH to 3.0 with H3PO4 and separating the precipitate by
centrifugation, the supernatant was extracted three times with peroxide-
free ether. The ether extracts were reduced to dryness, and the IAA
taken up in a small volume of water for colorimetric analysis using
Salkowski reagent.

The amount of fraction A obtained was 3.8 g per kilogram of corn
kernels in the first batch extracted, and 2.3 g per kilogram in the
second. The first batch was extracted three times with aqueous acetone,
and the second batch only two times. This could account in part for the
difference, furthermore, the corn used in the second extraction was from
a different batch, probably harvested a year later. The IAA content of
fraction A from the first batch was 0.8 Z, and from the second 0.53 Z.

The purification of fraction A was difficult, due to its peculiar

properties, especially its insolubility in water, which excluded the

31

application of mahy of the generally used fractionation methods. When
fraction A was chromatographed on a column of Sephadex G-25 with Tris
buffer pH 8.0, 0.002 M in 50 Z acetone, a single peak was obtained,
which suggested a large molecule, probably with a molecular weight
higher than 3,000. The IAA content of fraction A after treatment with
Sephadex was approximately the same.

Fifty milligrams of fraction A was hydrolyzed successively with
increasing concentrations of sulfuric acid. The first hydrolysis was
in l N H2804 for 6 hours in a boiling water bath. The sample did not
dissolve completely and the clear solution was separated and neutralized
with BaCOB, the precipitate separated by centrifugation and the super-
natant solution lyophilized. The process was repeated with 4 N H2804

being used in the second hydrolysis, and 6 N H in the third hydroly—

2504
sis. A brown precipitate still remained after the third hydrolysis.

The first hydrolysis yielded 7.6 mg of a slightly yellow powder, the
second hydrolysis 11.1 mg of an almost white powder, and the third
hydrolysis 4.2 mg of a white powder. Samples of 0.5 mg of each hydroly-
zate was used for a paper chromatographic analysis. The samples did not
dissolve completely in 0.1 m1 of water and after stirring for a few
minutes, the undissolved material was discarded and the clear super-
natant used. Whatman #1 chromatographic paper, and n-butanol:acetic
acid:water, 5:1:2.2 as the developing solvent were used. One chromato-
gram was treated with silver nitrate and the other with ninhydrin. The
result, shown in Figure 2, was as follows:

'Agflga: There are two faint spots on the chromatogram of the first hydro-

lyzate, with R 0.11 and 0.21; the second hydrolyzate shows the same

f

spots, but fainter, and there are only traces of them in the third

32

 

 

 

 

 

 

 

 

 

 

R
f Rf
0.0 A 43" g A g 13__1 0.0
”0 O O “
"O O 0 § § :9)“
o o‘
b ‘1
0 O
0.5- O O-o.5
' o 0*
_ O O
O _
1.0
AgNO3 Ninhydrin 1'0

Figure 2. Diagram of the chromatograms of the first, second,
and third H2804 hydrolysates of fraction A.

33

hydrolyzate. These spots suggest the presence of reducing carbohydrates,
probably a monosaccharide at the higher Rf.
Ninhydrin: There are three ninhydrin-positive spots in the first hydro—

lyzate, one of them with the same R as one of the AgNO3-positive spots.

f
There are 11 spots in the second and third hydrolyzate, the color being
more intense in the third hydrolyzate. This indicates the presence of

amino acids. The nitrogen content of fraction A was 8 Z and was deter-

mined by a Micro-Kjeldhal method (94). These results suggest that

fraction A is, or contains, a glycoprotein.
D. Purification and partial characterization of fraction B.

(1) Purification of the crude fraction.

Fraction B is the aqueous supernatant solution obtained upon
concentration of the acetone fractions. When the pH of this solution
is adjusted to 12.0 with KOH, free IAA is released, which can be ex-
tracted by washing with ether after adjusting the pH to 3.0 with
H3PO4.

Fraction B was lyophilized and a white powder, with a very high
content of NaCl from the extraction procedure was obtained. The IAA
content was between 0.06 and 0.13 Z. The IAA-complexes were separated
from the NaCl by using a column of Sephadex G-25. The column was pre-
pared with 260 g of Sephadex, and had a diameter of 5 cm and a length
of 50 cm. The bed volume was about 1000 ml. Fraction B was chromato-
graphed on this column in batches of 150 m1 of a 25 Z solution. The
separation between the IAA-complexes and the NaCl was good, but the

IAA-containing compounds came out in the 1250 to 1600 ml fraction of

the eluant, immediately after the NaCl, which came out in the 800 to

34

1250 m1 fraction. This suggested that the IAA-complexes were rather
small molecules, and that the separation on Sephadex was not due to
molecular size, but rather due to adsorption. When two milliliters of
a solution of fraction B containing about 50 micrograms of IAA was
dialyzed against one liter of water for four hours, only traces of IAA
were found in the dialysis bag. After purification with Sephadex and
lyophilization, fraction B was a white and very light powder with an
IAA content of 13.5 to 15.2 Z. The recovery of this purification step
was 43 Z.

Fraction B gave a purple color with Ehrlich's reagent, but no
color was formed with Salkowski reagent, unless the sample was previously
hydrolyzed with alkali. At this stage of purification, fraction B is
readily soluble in water and insoluble in ether. When a solution of
this fraction was washed with ether, all the IAA-containing compounds
were found in the water phase.

When fraction B was chromatographed on paper using n-butanol:acetic
acid:water as developing solvent, it was shown that it contained at

least four components, with R of about 0.40 to 0.60, and there was

f
no free IAA present. IAA has an R of about 0.90 in this solvent, and

f
the four IAA-containing compounds seem to be more polar than IAA.
When fraction B was chromatographed using either isoprOpanol:
ammonia:water, 8:1:1, or n-butanol(sat.with water):ammonia, 7:1, the
IAA complexes were broken down and the only spot which could be detected
in the chromatograms after treatment with Ehrlich's reagent was due to
free IAA.

Treatment of fraction B with concentrated ammonia at room tempera-

ture overnight resulted in the complete breakdown of the IAA complexes,

35

with formation of free IAA and indoleacetamide. Upon chromatography in
three different solvents, using synthetic indoleacetamide as a standard,
it was estimated that more than 50 Z of the original compounds were
ammonolyzed, and the rest hydrolyzed. This result indicates that IAA

is bound to the other part of the complex molecule through the carboxyl
group in a linkage more labile than the amide bond.

Fraction B was assayed for biological activity in the Aygpg
coleoptile straight growth test and in the Cucumber root inhibition
test (115). The concentration of fraction B was expressed in terms of
its content of IAA, and on this basis had about 50 Z of the activity
of an authentic IAA standard. Thus, the complex is less active, per

mole of IAA, than is free IAA.

(2) Separation of fraction B into its components.

Various attempts to separate the components of fraction B using
column chromatography were unsuccessful. Sephadex G—25, Whatman
cellulose powder, MN 300 cellulose powder, and finely ground Whatman
#3 chromatographic paper were tried, and water, ethanol-water mixtures,
and butanol-water mixtures were used as eluants. In all cases the IAA
containing material appeared in the eluant in a single broad peak.

A sample of 140 mg of fraction B, in solution in 2 m1 of water,
were chromatographed in a 1.1 cm x 45 cm column of Sephadex G—25, with
a bed volume of 45 ml. Water was used as eluant and the fractions
collected were analyzed by paper chromatography. It was found that
the first part of the single broad peak obtained was richer in the com-

ponents with the lower R on paper. Many attempts to get a better re-

f

solution were not successful. The fractions richer in the components

with the higher R on paper were collected separately and lyophilized,

f

36

and the same was done with the fractions containing mainly the components
with the low Rf in paper. The IAA content of the mixture of components
with the higher Rf on paper was 29.2 Z, while the mixture of components
with the low Rf contained only 15.2 Z IAA. This suggests that the
migration of the components of fraction B during chromatography with the
butanol-water solvent depends on the IAA content. In other words,
migration depends on the size of the residue attached to the IAA.

Since paper chromatography seemed to be the only method which gave
a good separation of the components of fraction B, a survey was made of
the different types of chromatographic paper and different solvents, in
an attempt to improve the method. The best result was obtained with the
system originally used, that is, n-butanol:acetic acid:water, 5:1:2.2
and Whatman #1 paper.

In preliminary experiments it was observed that when the components
of fraction B were eluted from the chromatograms, they were heavily
contaminated with UV absorbing substances. When a blank was analyzed
by eluting chromatographic paper with water, considerable absorption
was found between 200 and 300 millimicrons. When a 46 cm x 57 cm sheet
of Whatman #1 was cut into small pieces, soaked in water overnight, and
the eluant evaporated to dryness, 14 mg of a yellow powder, which gave
a color reaction with anthrone reagent, was obtained. Several methods
were tried to wash the sheets of paper free of this contamination. It
was considered acceptable for the existing conditions, to wash the
sheets of Whatman #1 with distilled water in a chromatography cabinet
for 60 to 70 hours. No bacterial or fungal growth was apparent during

the process.

A sample of 840 mg of fraction B, with an IAA content of 15.2 Z

37

was chromatographed on 42 sheets of washed Whatman #1 paper, using 20 mg
of sample per sheet. The chromatograms were develOped for 24 hours with
n-butanol:acetic acid:water. After this period of time the component

with the highest R was near the front of the paper. The location of

f
the desired compounds was accomplished by cutting narrow strips from
the center and both sides of the chromatograms and treating them with

Ehrlich reagent. The components were numbered from 1 to 4, B being

1
the component with the highest Rf, and B4 the component with the lowest
Rf. The areas of the chromatograms containing the different components
were cut out, out into small pieces, and eluted with water. Components
B1 and B2 are very close and the area where they are almost in contact
was eluted separately and rechromatographed in order to avoid cross-
contamination. The chopped paper was eluted three times for two hours
with distilled water and the eluants lyophilized.

The fractions were checked for contamination with the neighboring
components using paper chromatography. The dry samples were white and
light powders. The yields and IAA content of each is shown in table 1.

Since the IAA content of all the fractions was lower than the IAA
content of the original fraction B, they were obviously contaminated with
some material from the chromatographic paper. When 40 to 50 mg of the
samples were suspended in 1 m1 of water, a clear deep yellow solution
and a grey—brownish precipitate resulted. The precipitate was removed
by centrifugation and discarded. The supernatant fluid was chromato—
graphed on a column prepared with 20 g of Sephadex G—25, 1.5 cm x 54 cm,
and with a bed volume of 100 ml, in batches of about 40 to 50 mg in

solution in 1 m1 of water. The yellow colored material came out of the

column ahead of the IAA-complexes, but traces of yellow color were pre-

38
sent in the eluant containing the IAA-complexes. The IAA content of

the four components and the yield obtained are shown in table 1.

Table l. Fractionation and purification of the water soluble
IAA complexes from 15 kg of sweet corn kernels.

 

 

 

Fraction. Drygweight. IAA content. Total IAA.
B 840 mg 15.2 Z 126 mg
pager
chromatigraphy
B1 120 mg 15.0 Z
B2 108 mg 15.5 Z
B3 80 mg 11.5 Z
B4 86 mg 9.5 Z
394 mg 52 mg
Sephadei G-25
B1 56 mg 34.0 Z
B2 50 mg 26.2 Z
B3 42 mg 20.5 Z
B4 36 mg 22.2 Z
184 mg 48 mg

 

(3) Partial characterization of the components of fraction B.

a.- Ultraviolet spectra.

The ultraviolet spectra of the four components differ from the
ultraviolet spectrum of IAA. All of them show a broad peak of absorption
at 320 millimicrons and much more absorption at 235 millimicrons than
IAA. The absorption at 280 millimicrons is much more than could be

accounted for by the IAA contained in them. The spectra of B1 and B2

are more similar to IAA than the spectra of B3 and B4, but the peaks

at 288 and 274 millimicrons are sharper. B3 in addition, has a broad

peak at 264, and B two peaks at 272 and 256 millimicrons.

4

A sample of B contaminated with a larger amount of the yellow

1

O.D.

39

 

 

 

 

 

 

,
B
l , 32
r
l I l l l l l l 4 1] £411 1 1 l 1 LJ
B3 B4
ILLLI t l l 1 l A ll 1 P141 L IL I I 1 l A.
250 300 350 250 300 350
millimicrons millimicrons 1

Figure 3. Ultraviolet spectra of the four components of the
water soluble fraction B.

40

colored material mentioned above, shows fluorescence when observed under
ultraviolet light on a chromatogram. When the sample is hydrolyzed with
alkali and then chromatographed, the fluorescent spot is found again

at the same Rf as B1, even though the IAA-complex has been completely
broken down and all the material giving a color reaction with Ehrlich's
reagent is present now at the Rf of free IAA. The fluorescent Spot was
eluted and its ultraviolet spectrum showed two broad peaks at 320 and
295 millimicrons. These results suggest thepresence of a contaminant
with the same Rf as B1 and a high extinction coefficient. A similar
contaminant seems to be present in all four fractions. It probably
came from the chomatographic filter paper and probably accounts for

the absorption at 320 millimicrons and the increase in the absorption

at 290 millimicrons.

b. Determination of amino acids.

Samples of 200 micrograms of B B3, and B were hydrolyzed

1’ Bz’ 4
with 6 N HCl at 1000C for 12 hours. When a paper chromatogram of the

hydrolyzate was sprayed with ninhydrin, no colored spots appeared.

c. Determination of phosphorus.

No phosphorus was found when B B and B were hydrolyzed

1’ 32’ 3 4

with 3 N HCl at 100°C for 6 hours and analyzed with ammonium molybdate
reagent. The analysis was repeated by igniting the samples in the
presence of KOH and analyzing with the ammonium molybdate reagent, and
a negative result was also obtained. Glucose-l-phosphate was used as a
standard.

d. Determination of reducing groups.

The four components of fraction B give a faint gray color when

41

treated with silver nitrate in a paper chromatogram. Since this method
involves spraying with a NaOH solution, hydrolysis of the complexes
could occur. Furthermore, IAA itself gives a faint grey color with this
reagent. For these reasons, two non—alkaline reagents for reducing groups
were used: aniline phthalate in aqueous butanol, and aniline oxalate in
water (70, 111). A sample of 100 micrograms of each of the components
was spotted on filter paper, and 25 micrograms of glucose, fructose,

and arabinose, and 50 micrograms of cellobiose used as standards. After
spraying and heating at 1050C, there was a strong positive reaction in
all the standards, but no color was obtained with the samples. This
result indicates that there are no reducing groups in the intact mole-
cules of the IAA-complexes.

Samples of 100 micrograms of B B B3, and B were hydrolyzed

1’ 2’ 4
with NH40H, spotted on filter paper, and treated with silver nitrate,
aniline phthalate, aniline oxalate, and p—anisidine. The four complexes
gave a black spot with silver nitrate, but no color was formed with any
of the other reagents. This indicates that the IAA in the IAA—complexes

is attached to a group which gives a strong positive reaction with

silver nitrate.

e. Hydrolysis with ammonium hydroxide.
In order to simplify the nomenclature, the IAA complexes have been
called IAA-X, X being the unknown moiety bound to the IAA. According

to this B is equal to IAA-X B is equal to IAA-X2, and so on.

1 l’ 2

Samples of 0.1 ml of solutions, containing 100 micrograms of the
complexes, were hydrolyzed by addition of one drOp of concentrated
NHAOH and heating at 60°C for a few minutes. The hydrolyzed samples

were chromatographed using Whatman #1 paper and butanol-acetic acid-water,

42

 

 

 

 

R _
f it 31 132 E; Bf. IAf‘ IIllAe Rf
0.0 r v v r ' ' ' 0.0

O O l

r © 0 O
r .1
(9

t @ A
o 5 ~ ‘0 5

I- _

o o o o O

P O O O O O ‘

1.0 1.0

 

Figure 4. Diagram of a chromatogram of the four components of
the water soluble fraction, B , B , B , and B ,
hydrolyzed with NH OH. Standards are: C = cellobiose;
G = glucose; A = arabinose.

43

and the chromatograms treated with silver nitrate. Cellobiose, glucose,
and arabinose were used as standards. A diagram of the chromatogram is
shown in figure 4.

The R of X and X is the same, 0.12, and slightly higher than the

f 1 2
Rf of the Cellobiose standard. The Rf of both X3 and X4 is the same,
0.076, but much lower than the Rf of X1 and X2. In order to see if
there was some small difference between the migration of X and X , and

1 2

between the migration of X and X4, the four hydrolyzed samples were

3
chromatographed again using the same solvent, but developing the

chromatogram for 60 flours. The distribution of the spots and their

position with respect to the standards was the same.

f. Electrophoretic analysis.

The carbohydrate nature of the X moieties was suggested by the
chromatographic behaviour reported above. In order to obtain more
evidence concerning this possibility, the behaviour of the four com-
plexes during electrOphoresis in borate buffer was studied.

Samples of 50 micrograms of each fraction on Whatman #1 paper were
used for electrophoresis in 0.05 M borate buffer, pH 9.4, at 600 volts
for 2 hours. The potential gradient was 11 volts/cm. The paper strip
was treated with silver nitrate, and faint grey spots were obtained,
showing the migration of the compounds toward the positive pole.

The electrophoresis was repeated under the same conditions but for a
longer time, 4.5 hours, and the paper strip was treated with Ehrlich
reagent. Single spots and the same rate of migration was obtained for
all the four complexes, indicating that there was no breakdown of the
IAA-X molecules during the process. The IAA standard used migrates much

faster than the IAA-complexes, as it is shown in figure 5. In order to

44

 

 

 

 

(—) (—)
IAA B1 B2 B3 B4 IAA B1 B2 33 B4
0 o o o
o o
Gd 99

 

 

 

 

 

Figure 5. Diagram of the electrophoretograms of the four
components of the water soluble fraction, B , B ,

B , and B4 in: a) borate buffer pH 9.4; b) glygine
buffer pH 9.4.

45

determine if the migration of the IAA-X molecules was due to the forma-
tion of a charged complex with borate, the electrophoresis was repeated
using 0.05 M glycine buffer, pH 9.4, instead of borate buffer. There
was no migration of any of the four fractions. The rate of migration

of the IAA standard was the same as in borate buffer.

g. Hydrolysis with nitric acid.

In all the experiments involving acid hydrolysis, the IAA moiety
of the IAA—X complexes was destroyed, as would be expected. The com-
pounds detected on the chromatograms were the corresponding X part of
the molecules and their hydrolysis products.

A sample of 50 micrograms of B was hydrolyzed with 3 Z HNO in

3
a boiling water bath for 0.5, l, 2, and 4 hours. The samples were dried

l

and chromatographed, and the chromatogram treated with silver nitrate.
There was little hydrolysis of the X moiety, with formation of very

small quantities of a compound with R similar to glucose. However

f

most of the sample remained unhydrolyzed, and apparently there was no
more hydrolysis after 4 hours than after 0.5 hours. The small amount

of glucose could have resulted from a slight contamination of B1 with

B2.
h. Hydrolysis with hydrochloric acid.

Samples of 25 micrograms of B B B3, and B were hydrolyzed with

1’ 2’ 4
HCl at two concentrations, 1 N and 2 N, at 80°C for 6, 12, 24, and 48
hours, in sealed tubes. After hydrolysis the samples were dried,
chromatographed, and treated with silver nitrate for detection of the
spots. Very little hydrolysis of the X moieties was observed in all

the treatments. There are traces of a compound with the R of glucose

f

46

in all the samples, and some traces, more abundant in B4, of a compound

with the Rf of arabinose. X3 and X4 have the same Rf as X1 or X2 after

hydrolysis, suggesting partial hydrolysis of those compounds with con—

or X

version to compounds similar to X1

2.
i. Hydrolysis with sulfuric acid.

Samples of 30 micrograms of B B B3, and B were hydrolyzed

1’ 2’ 4

with sulfuric acid at two concentrations, 2 N and 4 N, at 800C for 6,
12, 24, and 48 hours, in sealed tubes. After hydrolysis the samples

were neutralized with BaCO the precipitate separated, and the samples

3’

chromatographed. Silver nitrate was used for the detection of the
spots.
There is very little hydrolysis of X1, with only traces of two

spots with the R of glucose and arabinose. Hydrolysis products are

f

present in much larger quantity in B but most of the sample is present

2’

as X2. There is a spot with the R

with the Rf of arabinose. There are two spots present in B3 and B4,

one with the Rf of glucose and the other with the Rf of arabinose. X3

and X4 have been converted to compounds with the same Rf as X1 or X2.

All the treatments gave about the same extent of hydrolysis.

f of glucose, and traces of a spot

Apparently there is no difference between treatment with 2 N sulfuric
acid or 6 hours and treatment with 4 N sulfuric acid for 48 hours in
any of the four samples. Synthetic IAA was hydrolyzed as a control and
no Spots were obtained, since probably complete decomposition occurred.
The results of this experiment suggest that there is a glucose-
like residue in X which is easy to separate from the rest of the mole-

2

cule. A glucose-like, and an arabinose—like residue seem to be easily

47
separated in X3 and X4, with conversion of these moieties to X1. X1

in turn seems to be very hard to hydrolyze.

j. Hydrolysis with HCl under mild and strong conditions.

In order to check the hypothesis advanced above about the behaviour
of the X moieties on acid hydrolysis, these were hydrolyzed under very
mild conditions: 1 N HCl at room temperature for 3 hours; and under
strong conditions: concentrated HCl at 1000C for 4 hours. All the sam-

ples were previously hydrolyzed with NH OH to separate the IAA from the

4

X moieties, and the NH OH was eliminated before the acid hydrolysis by

4

concentrating to dryness. The acid hydrolyzed samples were chromato-

graphed and AgNO was used for the detection of the compounds. Samples

3

hydrolyzed with ammonia but not with acid were used as controls. The
results are shown in figure 6.

The position of X does not change, and apparently there is very

1

little hydrolysis with the strong treatment. There are traces of mono—

saccharides present only in the mild treatment. The hydrolysis of X2

shows a similar result. Both X3 and X4 are partially converted to a

compound with the same Rf of X1 with the mild treatment, with simultan-

eous formation of two compounds with the same R of glucose and arabinose.

f

The strong treatment produces the total conversion of x; and X4 to the

Xl-like compound, but there are no traces of monosacharide—like com-

pounds, which are presumably destroyed.

The results of this experiment seem to confirm that the X moiety

1

of B1, is very resistant to acid hydrolysis. This X1 moiety is probably

identical to the compounds arising upon very mild acid hydrolysis of

X2, X3, and X4.

48

 

 

 

 

‘ x 5 x . x E x
Stgcslbls 921259 2312isé'12 f
0.0 3 E 3 ' V l v v '“1 v .7 *v E v 'v - 0.0
i i :0 O 300
”@iOOOiOOOE 00! 007
0.2— _ é a i ~04
©§ '13' i Q E O i Q
@g 0 .1; = Q 5 Q
I i i
— E l 5 i 4
i a i
: g . .
Figure 6.

Diagram of chromatogram of X , X , X3, and X (ob-

tained by alkaline hydrolysis of B

c =
b =
are:

, B , B3’ and B4)
control; a= hydrolyzed with 1 N HCI at room temp.;
hydrolyzed with cone. HCl at 1000C. Standards

C - cellobiose; G = glucose; A = arabinose.

49

k. Hydrolysis with 2.5 N sulfuric acid in an autoclave.

Fractions of 1 mg of each compound were hydrolyzed with 2.5 N
sulfuric acid in the autoclave at 15 psi. (about 120°C) for 1.5 hours.
After hydrolysis the solutions are brownish and there is some black
precipitate. The solutions were neutralized by addition of a small
amount of Dowex anion exchange resin in carbonate form and were kept
at room temperature until a neutral solution was obtained. The samples
were concentrated to dryness and chromatographed on paper for 24 hours
using ethyl acetate: pyridine: water, 8:2:1 as the developing solvent.
Galactose, glucose, mannose, arabinose, and xylose were used as
standards, and the chromatograms were treated with silver nitrate,
aniline phthalate, and p-anisidine. Silver nitrate gave much neater
spots than the other two reagents. Diagrams of the chromatograms are
shown in figure 7.

Both B and B yield a compound with the R of glucose upon hydro-

l 2 f
lysis. In B3 and B4 the hydrolysis products are two compounds with the
R of glucose and arabinose. The spots with the R of glucose and arabi-

f f

nose give the same color as the glucose and arabinose standards with
aniline phthalate and p-anisidine.

The unhydrolyzed compounds or the X moieties are not detected on
these chromatograms. In order to find out if this was due to total hydro-
lysis or to partial destruction of the compounds, the hydrolysis was
repeated for one hour under the same conditions, and the sames chromato-
graphed using n-butanol-acetic acid-water as the developing solvent.

The upper part of the chromatogram, from the origin to R 0.33 was

f

treated with silver nitrate, and the lower part was treated with Ehrlich's

reagent. The hydrolysis products are the same as before and the glucose

Figure 7.

50

 

 

 

 

Diagram of a chromatogram of the four components
of the water soluble fraction, B1, B , B3, and 34’
hydrolyzed with 2.5 N H 804 in autoclave at 15 psi
for 1.5 hours. Standards are: Ga = galactose;

G1 - glucose; M = mannose; A = arabinose; X = xylose.

51

and arabinose spots are very neat and have the same R as the glucose

f
and arabinose standards. No Ehrlich positive compounds were found in
the lower part of the chromatogram, and only some very diffuse spots at
the Rf of the X moieties in the upper part. This suggests that there is
partial destruction of the compounds during hydrolysis. The amount of

glucose obtained from B was very little, less than the amount produced

1
upon hydrolysis of the other fractions.

1. Hydrolysis with betaeglucosidase.

The fractions were incubated with beta-glucosidase in 0.05 M
acetate buffer pH 5.0 and at 300C, for 20 hours. Although there was
evidence of some hydrolysis, no conclusions were possible because the
enzyme preparation was contaminated with carbohydrate material. When
the enzyme was incubated without substrate and chromatographed, a small
amount of glucose and arabinose were obtained. The enzyme was purified
with Sephadex G-25, after incubation at 30°C for 6 hours to get hydro-
lysis of polysaccharide materials that could be present. When the sam-
ples were incubated with the purified enzyme, and with beta-glucosidase
from a different source, the result was better, but there was still some
carbohydrate material from the enzyme preparation in the chromatogram,
interfering with the detection of the hydrolysis products and the method
was discarded.

In one of the experiments with beta-glucosidase, fraction B1 was
incubated with buffer, as a blank, for 7 days. After this period of
time there was no decomposition of B1'
m. Hydrolysis with snail gut juice.

A sample of 1 ml of snail gut juice was diluted to 3 ml with

0.2 M phosphate buffer pH 6.0. The solution was centrifuged and the

52

precipitate discarded. The supernatant is a dark brown solution con-
taining about 10 mg dry weight per m1. A solution of 1 ml of each
fraction containing 1 mg of the IAA-complexes was incubated with 0.02
ml of the snail gut juice preparation for 48 hours at 30°C. After
incubation, the tubes with the reaction mixtures were heated in boiling
water for 5 minutes and stored in a freezer. The samples were chromato-
graphed on paper using butanol-acetic acid-water as the develOping
solvent. The upper part of the chromatogram was treated with silver
nitrate, and the lower part with Ehrlich's reagent. A diagram of

the chromatogram is shown in figure 8.

The results of this experiment agree with the results of the acid
hydrolysis. Since there is no breakdown of the IAA or the other units
of the complexes, the chromatograms are very neat and it is possible to
follow the change in the B fractions during hydrolysis by the change in
position of the Ehrlich positive spot in the chromatograms.

There is a small amount of glucose produced from B1, and much more
from B B3, and B4. Arabinose is present only in B and B . The R of

2’ 3 4 f

Bl has not changed, and there is no clear change in B2, but B3 and B4

are converted, upon release of the monosaccharide units, to a compound

with the same Rf of B1. There is no free IAA in the chromatogram,

indicating that the bond between the IAA and the X moieties is not

3 4

controls show two spots each, suggesting partial interconversion, from

affected by the incubation with the enzyme preparation. The B and B

B3 to B4 and, peculiarly, vice versa. Something similar could have

happended in B and B2, which appear in the chromatogram with the same

1

R In addition, the spots are elongated and shaped a little like the

f.

figure 8, suggesting two components.

53

 

 

A NO
g 3

r I
‘_ 0000 0 O :05

EHRLICH

 

 

 

1.0

Figure 8. Diagram of a chromatogram of the four components of
the water soluble fraction, B , B , B3, and B4,
hydrolyzed with snail gut juice. c = control,

E - incubated with snail gut juice. Standards are:
G = glucose; A = arabinose.

54

n. Quantitative estimation of glucose.

There was evidence from paper chromatography that there was
probably more glucose in the enzymatically hydrolyzed samples than in
those that were acid hydrolyzed. Several attempts were made to estimate
the amount of glucose in the hydrolyzate by using the Anthrone method,
but the results were inconsistent. The Glucostat reagent, which uses
glucose oxidase, is very sensitive and specific, but gave inconsistent
results with the enzymatic hydrolyzate. In all analyses it was found
that considerably less glucose was released from B than from the other

1

three fractions. After hydrolysis with 2.5 N H2804 in an autoclave for
1 hour, the amount of free glucose found in the hydrolyzate was about

8 Z in B1’ and 25 Z in B B3, and B

2’ 4'
0. Determination of inositol.

The X moieties obtained upon hydrolysis of the water soluble frac-
tions do not give a positive reaction for reducing sugars but react
strongly with silver nitrate and form a complex with borate, suggesting
the presence of a polyhydroxy compound which is not a sugar. The
possibility that the X moieties contained an hexitol was investigated.

B1 was hydrolyzed with ammonium hydroxide and chromatographed on
paper, with myo-inositol as a standard, and using four different sol-
vents: n-butanol:acetic acid:water, 5:1.5:3.5; isopropanol:acetic acid:
water, 5:1:7.5 (ascending); ethyl acetate:pyridine:water, 8:2:1; and
phenol:water, 4:1.

As is shown in figure 9, X1 and inositol have the same Rf in the

four solvent systems used.

A sample of 1 mg of fraction B was hydrolyzed with Rexyn AG4, a

1

quaternary amine strong anion exchanger, by adding resin in small amounts

 

 

0H
0%

 

{DI—I

 

 

55

 

’><

 

Rf

 

 

G

a-.-
“."

Gm

 

 

 

‘—- -----’

0.5

 

 

 

 

 

r
0.5"
r‘\
h- ‘-’
"‘t
I. i: ('
1.0
Figure 9.

 

 

 

Diagram of chromatograms of X and inositol.

{1.0793

= n-butanol-acetic acid-water.

= isopropanol-acetic acid—water.
ethyl acetate-pyridine-water.
phenol-water.

1.0

56

 

I Xl I+X

 

 

(H

 

 

Figure 10. Diagram of an electrophoretogram of X and
inositol, in borate buffer pH 9.2 and 0.05 M.

57

to a solution of B1 until the pH was constant at about 10.5. The hydroly-

zate was analyzed for inositol by the Scherer test described by Feigl and
Gentil (39). Three replicates of the sample and two standards of 25 and
50 micrograms of inositol were used. A pink color,indicating the pre-
sence of inositol, was obtained. A sample of the hydrolyzate was used
for electrophoresis in borate buffer pH 9.2 and 0.05 M, at 600 volts for
four hours, with a potential gradient of 11 volt/cm. A standard of
myOfimDSitol was used and the electr0phoretogram was treated with silver
nitrate. X has the same mobility as myo-inositol and when myo-inositol

1

and X1 were used a single spot was obtained, as shown in figure 10.

A sample of 10 mg of B was hydrolyzed by treating it with Bio Rad

l
AG-l X10, a strong anion exchange resin, in a small column (bed volume
about 0.5 ml). No IAA was detected in the eluant, which, after ly0phili-
zation yielded 1.1 mg of a white substance. The melting point of this
fraction and myo-inositol was determined. A small amount of X1, a small
amount of myo-inositol, and a mixture of both were used. A tan color

appeared in the sample of X at about 175°C, turning light brown at

1

200°C. The same was observed in the mixture of X1 and myo-inositol.

The melting point of myo-inositol was 224-2250C, the melting point of

X1 was 221-2230C, and the mixed melting point was 221-2250C. Upon
cooling, the samples recrystallized and the melting point was determined
again, obtaining the same values.

The results of these analysis give good evidence of the presence

of myo-inositol in X1.

DISCUSSION

 

During the last three decades indoleacetic acid has been regarded
as a plant growth hormone, and its participation in growth and morpho-
genesis in plants has been well established. However, although it has
been generally accepted for many years that free indoleacetic acid is
the active form of the auxin, there is actually no conclusive evidence
to support this idea, and there is now a growing body of evidence which
points to the importance of the bound forms. There are authors as, for
example, Bentley (21) who think that the free form of IAA is not the
auxin physiologically active in normal growth.

There has been a great deal of confusion in this field, owing
mainly to the use of inadequate techniques of extraction and purification,
and inadequate chemical characterization. Extraction of plant tissues
with water or solvents that do not inactivate enzymes might lead to the
‘breakdown of labile forms during the process. Even the solvent itself,
as in the case of ethanol under alkaline conditions, can give rise to
artifacts (165). Solvents containing ammonia have been used extensively
in chromatography of indole compounds, because they give a good resolu-
tion for several simple indole derivatives, but the use of this method
results in the breakdown of labile complex forms of IAA during the pro-
cess. The use of ammoniacal solvents, as 130prcpanol-ammonia-water, has
been responsible for the report of artifacts in a very large number of
papers. In 1961 it was clearly shown by Zenk (163) that the indoleaceta-
mide and the indoleacetic acid he found in chromatograms of plant ex-
tracts, were artifacts due to the use of ammonia in the developing sol-
vent. In spite of this, there are still some authors who rely on this

method for the analysis of plant extracts. Diffusion techniques, such

58

59

as diffusion from coleoptile tips into agar undoubtedly permit hy-
drolytic enzymes liberated at the cut surface to act.

Inadequate chemical characterization has been very common, and
actually it is possible to say that only in a few instances has a
careful characterization been tried. Many reports are based upon
bioassay of crude extracts or upon bioassay, or chemical analysis, of
crude fractions separated by a single chromatographic step.

Recently it has become evident that several of the derivatives
with growth regulatory activity which have been reported, are breakdown
products of more complex molecules. Glucobrassicin, recently reported
as the principal indole compound in cabbage (83) has been found to give
rise to a number of indole derivatives upon enzymatic and chemical
hydrolysis. Some of the degradation products of glucobrassicin are
(44): ascorbigen, indole-3-acetonitrile, 3-hydroxymethylindole,
indoleacetamide, indoleacetic acid, indolecarboxylic acid, skatole, and
indole. The sensitivity of glucobrassicin to heat or hydrolytic condi-
tions and its spontaneous cleavage by myrosinase, which is present in
cabbage tissues, explain the presence of some of these "artifacts" in
extracts of cabbage. The fact that some of the degradation products of
glucobrassicin are products of a reaction catalyzed by an enzyme present
in cabbage tissue, indicates that some of them could be considered as
naturally occurring compounds.

Apparently glucobrassicin has been reported only in Brassica, but
this, or similar compounds, could be present in other plants. The
possibility exists that IAA is present in plant tissues in a few bound,
and very labile, forms, which could be the key substances for the

hitherto described indole derivatives, including IAA, which have been

60

reported in plant tissues. The variety of indole derivatives which have
been found could arise from these compounds. Different methods of
extraction and purification could give rise to different derivatives.

Fraction A, the water insoluble fraction reported in this work,
has been demonstrated to be a high molecular weight compound, similar
in its physical pr0perties to the fraction isolated by Berger and Avery
(22), but with a lower content of IAA and a higher content of nitrogen.
The fraction reported by Berger and Aver had 3.7 Z IAA in its purest
form, and 4.7 Z nitrogen. Fraction A has between 0.5 and 1.0 Z of IAA,
and about 8 Z nitrogen.

The isolation and partial characterization of the bound forms of
IAA was restricted to two samples of corn of the same variety, but
obtained in different years. This could explain, in part, some of the
differences found between the fractions obtained from the two samples.
The first sample, of 6 kg, was extracted three times with aqueous
acetone, and the second sample, of 15 kg, was extracted only two times.
The yield obtained in the first case was 3.78 g of fraction A, with
0.8 Z of IAA, per kilogram of corn, this being equal to 30 mg of IAA in
the water insoluble form per kilogram of corn. The yield from the sec-
ond sample was 2.28 grams of fraction A, with 0.53 Z IAA, per kilogram
of corn, equal to 15 mg bound IAA in water insoluble form per kilogram
of corn. The quantity of water soluble IAA complexes was larger in the
second case than in the first, being equal to about 15 mg of IAA per
kilogram, and to about 20 mg of IAA per kilogram of corn in the second.
Thus, the total amount of bound IAA obtained was equal to about 45 mg
per kilogram of corn in the first sample, and to 32 mg per kilogram in

the second. If however IAA content is calculated as percentage of the

61

total weight of the complexes including water soluble and insoluble forms
(weight of fraction A plus fraction B) the value obtained for the two
samples is not very different, being equal to 1.2 Z for the first, and
1.35 Z for the second.

The peculiar solubility properties of fraction A made its purifi-
cation difficult. Fraction A, like zein, is soluble at pH 11—12, but
under these conditions, there is hydrolysis of the IAA—complex, with
release of IAA. The fact that fraction A appeared in a single peak in
the eluant after chromatography in Sephadex G—25, and with about the
same content of IAA, does not indicate that the compound is pure, since
it could be contaminated with high molecular weight compounds, which
are not separated from the IAA—complex during gel filtration. Apparently
zein has at least three components with different molecular weight (45)
and the same could be true for fraction A. The main obstacle in the
purification of this fraction was the lack of methods suitable for com-
pounds with these solubility characteristics

Zein is almost certainly associated with fraction A. As was ex-
plained in the Introduction, there seems to be a correlation between the
disappearance of water soluble forms of IAA and synthesis of zein in
maturing corn kernels. Zein is a peculiar protein, this type of pro-
tein being present mainly in cereal seeds. It is probably notlikely
that bound forms of IAA similar to fraction A are very widely distributed
in plants. Apparently the only fraction reported in the literature simi-
lar to the water insoluble fraction A found in this work is the "auxin
precursor" described by Berger and Avery (22), and the fraction reported
by Hamilton (58), extracted with 80 Z ethanol from corn kernels.

The purification of the water soluble complexes was achieved by

62

paper and Sephadex G-25 chromatography. The four components isolated
have very similar solubility pr0perties and this makes their separation
difficult, since they run very close to each other on paper chromato-
graphy, and on different adsorbents in column chromatography. A great
deal of time was spent trying to find a suitable column chromatographic
method to separate these compounds, but without success. The method
used in this work permits a good separation, but the yield is poor,
about 40 Z, and there is a great deal of contaminating material coming
from the paper. Fifty large sheets (46 x 57 cm) of Whatman #1 paper
are necessary for the separation of the components of l g of fraction B,
and the process is very time consuming. A better method, probably based
on column chromatography, where larger amounts of material can be used
and no contamination from the adsorbent is obtained, should be worked
out.

The water soluble bound forms have not been completely characterized.
There is information about the units present in the complexes, and about
the IAA content of each of them, but the molar ratios of the different
components have not yet been obtained. The study of the composition of
the complexes was done by analyzing the hydrolysis products after alka-
line, acid, or enzymatic treatment. Treatment of the samples with con-
centrated ammonium hydroxide yielded indoleacetamide, showing that the
IAA is bound to the carbohydrate moiety through the carboxyl group.
Very mild alkaline treatment hydrolyzed the complexes by breaking the
bond between the IAA and the carbohydrate moiety, showing that the same
type of bond (very labile to alkali) is present in the four water
soluble fractions. In the water insoluble fraction A, IAA is linked to

the rest of the molecule by a similar alkali-labile bond.

63

A preparation of snail gut juice split the water soluble complexes
quantitatively and very neatly, presumably without breakdown of the
individual components. Since however a mixture of enzymes is present
in this crude preparation, no information was obtained about the nature
of the bonds between the units.

As a working hypothesis and as a guide to the reader, the present

concept of the composition and structure of these complexes is shown

in figure 11.

 

Bl (IAA - INOSITOL)){
B2 (IAA - INOSITOL - GLUCOSE))(
B3, B4 (IAA - INOSITOL - GLUCOSE - ARABINOSE))(

 

Figure 11. Tentative composition of the water soluble complexes.

The available evidence shows that B1 contains IAA and inositol.

When B1 was hydrolyzed with acid or enzyme, there was always a small

I
amount of glucose in the hydrolyzate. The Rf of B1 did not change when

treated with snail gut juice, and the R of X1 (Bl minus the IAA) did

f
not change when treated with acid. Something similar was observed when
82 was hydrolyzed, as is explained below. Inositol, and inositol-glucose
could have the same behaviour in paper chromatography and paper electro—
phoresis. The calculated IAA content for a molecule containing IAA,
inositol, and a six-carbon carbohydrate unit is 35.1 Z. The IAA con-
tent found in B is 34.0 Z. The small amount of glucose obtained from

1

B1 when hydrolyzed with acid or enzyme, indicated the possibility that

this compound could be formed only by IAA and one unit of inositol. In

64

that case, however, about 33 per cent of the weight of this sample
would be impurities.

B2 contains IAA, inositol, and glucose, but the amount of IAA

found in this complex was 26.2 Z. The theoretical content of IAA in a
compound containing IAA, inositol, glucose, and a six-carbons carbohy-
drate unit, is 26.5 Z. A much larger amount of glucose is obtained

from B upon hydrolysis than from B

2 When enzymatically hydrolyzed,

1.

B2 is apparently converted to B1’ with release of glucose. The

difference between B1 and B2 seems to be the presence of one more glu-

cose unit in B2. The Rf of X2 and X1 are the same, and the Rf of X2

does not change when treated with acid, in spite of the appearance of

glucose in the hydrolyzate, showing that the Rf of inositol linked to

at least one glucose unit is the same as the R of free inositol. If

f

the hypothesis about the composition of B1 and B2 is correct, it would

imply that the R of inositol and inositol-glucose is the same in the

f

solvent system used.

B3 and B4 contain IAA, inositol, glucose, and arabinose. The IAA

content is 20.5 Z in B3, and 22.2 Z in B They have a different R in

f

, and some evidence

40

paper chromatography, B being more polar than B

4 3

for interconversion between them was found. The theoretical IAA content
for a molecule containing IAA, inositol, glucose, arabinose, and a six

carbons carbohydrate unit, is 22.1 Z. B3 and B4 are quantitatively

converted to B1 by incubation with snail gut juice, with release of

glucose and arabinose. X3 and X4 have the same Rf

graphy, but different from the Rf of X1 and X2. Both X3 and X4 are

with mild acid hydrolysis, with release

in paper chromato-

quantitatively converted to X1

of glucose and arabinose.

65

In all the water soluble IAA complexes, the IAA is bound to ino-
sitol. This bond is very labile to alkali, but is not attacked by snail
gut juice or beta-glucosidase.

There is a very close relationship between the water soluble IAA

complexes. The hypothesis could be advanced that B is a conjugate of

l

IAA-inositol-glucose, plus X; B3 and 34 IAA-

inositol—glucose- arabinose, plus X. The difference between B3 and B4

IAA-inositol, plus X; B2

is not, as yet, clear.
The relative abundance of these forms is as follows: B1 39 Z,

3 17 Z, and B4 17 Z. This type of distribution would probably

be obtained if B1 is a "limit" product of the breakdown of a straight

chain as long as B3 or B4 or longer, with very labile bonds between the

B2 27 Z, B

units beyond the B moiety. There is a possbility that the water solu-

l
ble complexes were originally part of a high molecular weight compound
like fraction A. The presence of amino acids, and some reducing spots
with the Rf of monosaccharides and inositol after acid hydrolysis of
fraction A, and its content of nitrogen of about 8 Z, indicates that

it is a glycoprotein-type compound in which IAA could be bound to the
carbohydrate moiety, as it is in the water soluble fractions. There is
evidence that the bond linking the IAA to the rest of the molecule is
very similar in the water soluble and water insoluble fraction. Then,
the possibility exists that there was originally a single compound
whose components were IAA-inositol-glucose-glucose-arabinose-glycopro-
tein. A compound of this type, with labile glucose-glucose, glucose-
arabinose, and arabinose-glycoprotein bonds, could yield, upon partial

breakdown of these bonds, all the IAA-complexes found in this investiga-

tion. The straight chain structure was assumed for simplicity, but a

66

structure with inositol as a nucleus with the other components bound to
it, or other arrangements would be conceivable.
Fraction A is very similar to the water insoluble fraction ob-

tained by Berger and Avemr(22) using the same extraction procedure.
The main difference is in the content of IAA and nitrogen. Fraction A
has 0.53 Z IAA and 8 Z nitrogen. The fraction of Berger and Avery had
3.7 Z and 4.7 Z N. Different varieties of sweet corn were used and
that could explain the differences, but the fact that fraction A has a
higher content of nitrogen and a lower content of IAA, suggests that
there could be a correlation between IAA and N content. If we visualize
a model in which IAA is bound to the carbohydrate part of a glycoprotein,
a loss of carbohydrate chains with IAA attached to them, as in the
water soluble fractions described in this work, would increase the con—
tent of nitrogen and decrease the IAA content of the remaining glyco-
protein. In this work the IAA complexes were extracted with aqueous
acetone at room temperature, and enzymatic activity, which could break
up the original glycoprotein, is conceivable under those conditions.

The amount of IAA in bound form isolated during this work was up to
45 mg per kilogram of mature corn kernels. No estimation was made of
the total amount of bound IAA present in this material, but undoubtedly
the quantity actually present was larger. The weight of one kernel of
the variety used is about 200 mg, that is, there are about 5000 kernels
per kilogram. The amount of IAA in bound form in each kernel is then,
at least, about 10 micrograms. Hamilton (58) based on information
found in the literature, estimates that 10,000 oat coleoptile tips
might contain about 1 microgram of IAA. It is clear then, that the a-

mount of bound IAA found in sweet corn kernels is indeed a large amount

67

from the physiological point of view (about 100,000 times more than in
an Ayggg coleoptile tip) and more than enough to account for the pro—
motion of growth in the young seedling.

The crude fraction B assayed for biological activity was shown to
be active in the promotion of growth, although less active than IAA.
The fraction assayed was rather crude, and, as has been reported above,
not homogeneous and thus it is possible that not all the components _
are active. The purified components have not been tested for growth
promoting activity.

Hamilton (59) failed to find free IAA when he extracted, with
ether, an acidified 80 Z ethanol extract of growing (5 day old) corn
shoots, but about 13 micrograms per kilogram of fresh weight were found
when he first adjusted the pH to 8.0 for preliminary removal of neutral
ether soluble substances, and then acidified and extracted with ether.
He concluded that IAA could occur in growing corn shoots in the form
of a very labile complex, from which it was liberated by the alkaline
pH treatment. It should be emphasized that the alkaline treatment,
which was not intended for hydrolysis and is almost routine in the ex-
traction and purification of IAA, was extremely mild, and probably more
IAA could have been obtained by using a somewhat stronger alkaline
treatment. The complex suggested by Hamilton as present in growing
corn seedlings could be very similar to the water soluble fractions
found in this work.

Terpstra (138, 139) in a study of the extractable auxins from
.Ayggg coleoptiles, demonstrated that the auxin is present in extract
of ground coleoptile tips as water-soluble, ether-insoluble complexes,

which yield IAA very easily.

68

Many water soluble auxin complexes which yield IAA on hydrolysis
have been found to be naturally occurring (10, 27, 28, 32, 35, 36, 71),
but they have not been characterized. The "zeanins" found by Farrar
(35) in immature corn kernels have some similarities with the water
soluble fractions found in this work, as for example, similar behaviour
in paper chromatography in a butanol-water mixture and lability to
ammonia. The zeanins give a purple color with Salkowski reagent, and
this does not occur with the water soluble forms reported in this work,
which do not give a color reaction with Salkowski reagent, unless they
are first treated with alkali.

Farrar (35) considers the zeanins as intermediate compounds be-
tween native bound IAA and free IAA. It is not known yet if the water
soluble fractions found in this work are "artifacts" produced by break-
down of a larger water insoluble molecule, similar to fraction A, or if
they are normally present in corn as an intermediate form, as has been
suggested by Farrar for the zeanins.

The fluctuations in the auxin content in developing corn kernels
suggest that IAA is present, in early stages of the deve10pment of the
seed, in a water soluble bound form, which probably is gradually con-
verted to a high molecular weight, water insoluble form which is pre—
sent in the mature kernel. The "reserve materials" of the seeds, which
are largely macromolecules, are metabolically broken down during germ-
ination and utilized by the young seedlings. The fate of the large
amount of IAA present in corn, and probably other seeds, is not known.
A mechanism for germination could be visualized where an active form of
IAA were liberated from a glycoprotein—IAA complex by the action of

enzymes activated upon absorption of water by the seed.

69

Myo-inositol is widely distributed in microorganisms, plants, and
animals, and it is considered a growth factor for some microorganisms
and for warm-blooded animals. It is known that inositol participates
in some way in cell wall metabolism. Deficiency of inositol induces
agregation of yeast cells, and a greater proportion of glucan in the
cell wall (29). In a strain of Neurospora crassa inositol deficiency
gives rise to colonial growth (29). The mode by which inositol brings
about alterations in the polysaccharide composition of fungal cell walls
is unknown, but definite effects in polysaccharide biosynthesis have
been demonstrated. Inositol deficiency may also be induced by inositol
antimetabolites, with consequent effects on morphogenesis.' Myo-inositol
has been shown to be present in, and to be characteristic of, three
natural sources of the growth promoting ”neutral fraction" from nggg,
£23, and Aesculus (114).

It is not good scientific reasoning to assume that a conjugated
form of IAA and inositol is physiologically important in growth regula—
tion simply because it is present in large amounts. On the other hand,
it would be a great mistake to disregard the possibility of some such
role in view of the fact that both compounds are known to be involved
in plant growth and morphogenesis, and three decades of work have not
shown with any certainty that free IAA is present in plant tissues; the
amount of "free" IAA reported has often been so small that is presence
has been guessed rather than demonstrated.

The real problem to be faced now is to find out what is the active
form of auxin in plant tissues. Definitive evidence about the nature
of the native plant auxin will be difficult to obtain and will probably

require several kinds of experimental approaches. In addition to the

70

difficulties involved in identifying the forms present, due to the
labile nature of many of them which might lead to the production of
artifacts and mistaken identifications, there is the problem of finding
out what the physiological role of the naturally occurring forms is.
Zenk (165) has advanced the hypothesis that the action and formation

of IAA could be controlled by conjugation to form inactive compounds,
probably implying that free IAA is the active form. The presence in
animal tissues of conjugated forms of IAA which are considered detoxi-
fication products (74, 75, 127) and the fact that large amounts of
exogenous IAA are "neutralized” by conjugation in plant tissues (4, 6,
164) have pointed to the possibility that a similar system could be
operative in normal plant metabolism and give rise to bound forms. The
high concentration of bound forms found in some seeds suggests that
they are storage forms, which probably would be inactive as such.

It is not known how IAA is transported in the plant, but a low mole-
cular weight "neutral" compound, as a conjugate with sugars, would
probably be a suitable form.

Final identification of an active auxin complex as "native" will
most likely require an in vitro system which respons to the added
complex. If in addition the complex is ubiquitous in nature and occurs
in the right concentration in growing tissues, then it would probably
be safe to conclude that the naturally occurring active form of auxin

has been found.

CONCLUSIONS

Two indoleacetic acid containing fractions - one water-soluble and
one water-insoluble - have been isolated from mature sweet corn
kernels and partially characterized.

The water-soluble fraction was further fractionated into four com-
ponents: one containing indoleacetic acid and inositol; one con—
taining indoleacetic acid, inositol, and glucose, and two containing
indoleacetic acid, inositol, glucose, and arabinose. The indole-
acetic acid is released from these compounds upon very mild alkaline
hydrolysis.

The water-insoluble, aqueous acetone—soluble fraction, is a high
molecular weight compound, containing 0.5 to 0.8 Z indoleacetic

acid and about 8 Z nitrogen. This fraction yields indoleacetic

acid upon very mild alkaline hydrolysis, and amino acids, and pro-
bably sugars and inositol, upon acid hydrolysis.

There is a minimum of 45 mg of indoleacetic acid in bound form

per kilogram of mature sweet corn kernels.

The conjugation of indoleacetic acid with inositol is a new

finding and could have physiological importance.

71

10.

ll.

12.

13.

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