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This is to certify that the

dissertation entitled

The Presence and Transiocation of Indole-3-acety1-

m oginosttol in Shacts of Zg§_maxs L.

presented by

John R. Chisneil

has been accepted towards fulfillment
of the requirements for

Ph.D. Botany and Plant Pathology

degree in

 

 

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Major professor

Date q\\\o\8;73

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THE PRESENCE AND TRANSLOCATION 0F
INDOLE-3-ACETYL-filg-INOSITOL
IN SHOOTS 0F gag MAYS L.

By

John Richardson Chisnell

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1984

ABSTRACT

THE PRESENCE AND TRANSLOCATION or
INDOLE-3-ACETYL-MYO-INOSITOL
IN SHOOTS 0F.ZEE_MAYS L.
3v

John Richardson Chisnell

1. Enzymatic Synthesis of [5-3H]Indole-3-Acetic Acid and
[5-3H11ndole-3-Acetyl-myo-Inositol from L-[S-BHJTryptophan. High
specific actibity L-[5-3H1tryptophan is converted to [5-3H]indole-3-
acetic acid by enzymes from Pseudomonas savastanoi. [5-3H]indole-3-
acetic acid can then be converted to [5-3H]indole-3-acetyl-1-0-8-D-
glucose and subsequently to [5-3H]indole-3-acetyl-myg-inositol by
enzymes from kernels of Zgg_mgy§_sweet corn. Synthesis is on a nano-
molar scale with little or no dilution of the initial specific activity.
Yields, based on L-tryptophan, were 68% when [5-3H]indole-3-acetic acid
was the end product and 40% when [5-3H]indole-3-acetyl-myg-inositol was
the end product.

11. Myg-Inositolggsters of Indole-3-Acetic Acid are Endogenous
Components of Zea mays L. Shoot Tissue. Indole-3-acetyl-m o-inositol
esters have been demonstrated to be endogenous components of etiolated
Zea mays shoot tissue. This was accomplished by comparison of the
putative compounds with authentic, synthetic esters. The properties

compared were liquid and gas-liquid chromatographic retention times and

John Richardson Chisnell
the 70 eV mass spectral fragmentation pattern of the pentaacetyl
derivative. The amount of indole-3-acetylemyg-inositol esters in the
shoots was determined to be 74 nmol-kg'1 fresh weight as measured by
isotope dilution, accounting for 19% of the ester indole-3-acetic acid
of the shoot. This work is the first characterization of an ester
conjugate of indole-3-acetic acid from vegetative shoot tissue using
multiple chromatographic properties and mass spectral identification.
It is of interest that the kernel and the seedling shoot both contain
indole-3-acetylemyg-inositol esters, and that these esters comprise
approximately the same percentage of the total ester content of the
kernel and of the shoot. .

III. Translocation of Radiolabeled Indole-3-Acetic Acid and
Indole-B-Acetyl-my9-Inositol from Endosperm to Shoot of Zea mays L.
The distributions of total radioactivity, radiolabeled indole-B-acetic
acid and radiolabeled ester conjugated indole-3-acetic acid in the
shoots of dark-grown'ggaumays after the application of either
[5-3H1indoie-3-acetic acid or [5-3H1indoie-3-acetyi-m o-inositol to the
endosperm have been determinded. Sharp differences were found in the
distribution and chemical form of the radiolabeled indole-3-acetic acid
in the shoot depending whether [5-3H1indoie-3-acetic acid or
[5-3H1indole-3-acetylemygeinositol was applied to the endosperm. We
show that indole-3-acetylemyg—inositol applied to the endosperm
provides both free and ester conjugated indole-3-acetic acid to both the
mesocotyl and coleoptile. we calculate that indole-B-acetic acid in the
endosperm may supply some of the indole-3-acetic acid in the mesocotyl

but that it supplies essentially none to the coleoptile or primary leaves.

ACKNOWLEDGEMENTS

First of all I want to deeply thank Professor Robert S. Bandurski
for his support and guidance thoughout my graduate years. I also
wish to thank my committee members Professors Robert P. Scheffer
Kenneth D. Nadler, Derek T.A. Lamport, and Norman E. Good for their
advice and counsel. 1 am endebted to the many grad students, post docs
and technicians that I had the pleasure to associate with at Michigan
State University. I especially want to acknowledge Prudy Hall,
Roger Hangarter, and Leszek Michalczuk for their warm companionship
during my years at Michigan State University. Finally, I want to

thank Margo Daub for loving me throughout this graduate experience.

ii

TABLE OF CONTENTS

Page
LIST OF TABLES .......................... iv
LIST OF FIGURES ......................... v
INTRODUCTION ........................... 1
LITERATURE REVIEW ........................ 4
EXPERIMENTAL ...... . ................... 10
I. Enz tic Synthesis of [5-3H]Indole-3-acetic Acid and

[5- H31ndole-3-acetylgmyg-inositol from

L-[S- H]Tryptophan .................. 10

II. gygyinositol Esters of Indole-3-acetic Acid are
n ogenous Components of gga_mays L. Shoot Tissue . . 18

III. Translocation of Radiolabeled Indole-3-acetic Acid
and Indole-3-acetyl-m o-inositol from Endosperm

to Shoot of Zgg_mays . ............... 44
CONCLUSIONS ........................... 89
LITERATURE CITED ....... . .................. 92

LIST OF TABLES

EXPERIMENTAL II

I.

II.

III.

Characteristic ions in the 70 eV mass spectra of the
pentaacetyl derivatives of authentic IAInos and of

IAInos from ;, mays shoot tissue ...........

Quantitative analyses of IAInos, free IAA and free

+ ester IAA in ;, mays shoot tissue .........

The average and relative amounts of IAInos, free IAA

and free + ester IAA in ;, mays shoot tissue .....

EXPERIMENTAL III

I.

II.

III.

IV.

Raw data on total radioactivity in the shoot from
C

JIAA applied to the endosperm ..........

Raw data on free [3H]IAA and free + ester [3H]IAA in

the shoot from [3H]IAA applied to the endosperm . . .

Raw data on free [3H]IAA and free + ester [3H]IAA in

the shoot from [3H]IAInos applied at the endosperm . .

Raw data on the longitudinal distribution of free
[3H]IAA and free + ester [3H]IAA in the shoot

from [3H]IAInos applied to the endosperm .......

Raw data on the distribution of radioactivity in the
kernel after incubation with applied [3H]IAA and

[3H]IAInos ......................

iv

Page

37

39

75

76

77

LIST OF FIGURES

EXPERIMENTAL II

1.

3

TSD chromatograms of acetylated authentic IAInos
and of acetylated putative IAInos isolated from

;, mays shoot tissue .................

Tge GLC elution profiles of 3H from injections of
[ HJIAInos (A) and of putative [3H]IAInos reisolated

from Z. mays shoot tissue ..............

.. The 70 eV spectrum of pentaacetyl IAInos .......

EXPERIMENTAL III

1.
2.

Diagram of a 4-d-old dark grown £33 mays seedling

Location of the tissue sections used in the

longitudinal distribution experiments ........

The distribution of radioactivity in the shoot as a
function of time after application of [14C]IAA

to the endosperm ...................

The distribution of [3H]IAA, free and ester,3in the
shoot 2 h and 4 h after the application of [ H]IAA

and [3H]IAInos to the endosperm ...........

The concentration of [3H]IAA, free and ester, in the
shoot 2 h and 4 h after the application of

[3H]IAA and [3H]IAInos to the endosperm .......

The longitudinal distribution of radioactivity (A)
and concentrations of radioactivity (B) in the
shoot as a function of time after application of

[3H]IAInos to the endosperm .............

The longitudinal distribution of [3H]IAA, free and
ester, and total radioactivity in the shoot 4 h

after application of [3H]IAInos to the endosperm . . .

Page

41

43

83

87

Page
EXPERIMENTAL III, cont.
8. The longitudinal distribution of the concentrations
of [3H]IAA, free and ester, and total radioactivity

in the shoot 4 h after application of [3H]IAInos to
the endosperm .................... 88

vi

INTRODUCTION

It is well documented that the tip of a growing shoot plays a
special role in regulating the growth of the lower portion of the shoot
(Boysen-Jensen, 1936, Went and Thimann, 1937, Van Overbeek, 1939 A
Thimann, 1977). The studies of Went, Cholodny, Snow and others
indicated that the shoot tip supplied the growth hormone auxin
(presumably indole-3-acetic acid (IAA)) to the lower portion of the
shoot. However, the exact chemistry of IAA production by the tip is
unclear. It has not been established whether the tip is synthesizing
IAA g; 3919, or from tryptophan, or by hydrolysis of IAA conjugates.
This work is addressed to that question by studying one of the possible
precursors of IAA (IAA-m o-inositol) for conversion by the tip.

Early work on auxins by Cholodny (1935) and Van Overbeek (1941)
suggested that the seed was the source of an auxin precursor in young
seedlings. The structures and concentrations, down to a level of
10 ug-kg'l, of all the IAA containing compounds and the metabolic turn-
over rates of a number of the indolylic compounds in the kernel of Egg
mgy§_have been determined (cf. Cohen and Bandurski, 1982). In the
g;_mgy§_kernel IAA-m o-inositol accounts for approximately 30% of the
total IAA while free IAA accounts for only about 1% of the total IAA
(cf. Cohen and Bandurski, 1982). In the young dark-grown shoot of
;;_mgy§ ester conjugates of IAA constitute up to 80% of the total IAA

2

present (Bandurski and Schulze, 1977). Nowacki and Bandurski (1980)

demonstrated that exogenous radiolabeled IAA-m o-inositol was trans-

located from the endosperm to the shoot and that it was hydrolysed in
the shoot releasing free IAA. They also calculated that a sufficient
amount of ester IAA in the shoot was derived from IAA-m o-inositol in
the endosperm to potentially supply the estimated auxin needs of the

growing shoot.

At the time this study was initiated (78/79) there was evidence
that the seed is the source of an auxin precursor for the young dark-
grown seedling, that IAA-m o-inositol constitutes a significant amount
of the the low molecular weight esters of IAA in the ;;_mgy§ kernel,
and that IAA-m o-inositol can be translocated from the endosperm to the
shoot and there be hydrolyzed, releasing free IAA. Further, it had been
estimated that sufficient IAA-m o-inositol is translocated from the
endosperm to the shoot to potentially supply the auxin needs of the
growing shoot. This study then set out to further test the hypothesis
that IAA—mygfinositol is a seed auxin precursor in the shoots of dark-
grown g;_may§ by 1) establishing the natural occurrence of IAA-Exp:
inositol in the shoot; by 2) determining the route of transport of
IAA-mygyinositol from the endosperm to and through the shoot; and by
3) determining the pattern of distribution of IAA-m o-inositol and IAA,
derived from IAA-m o-inositol, in the shoot. In order to perform these
studies it was first necessary to synthesize high specific activity
tritium labeled IAA-_y97inositol. IAA-m o-inositol is not available
commercially, labeled or unlabeled. The tritium label was needed in
order to have a sufficiently high specific activity for its use in

tracer amounts. Tracer amounts were required 1) in order to be able to

3
detect the endogenous IAA-m o-inositol'hithe vegetative tissues, which
was expected to occur only at a low concentration, and 2) not to
significantly perturb the normal processes of translocation of IAA-mygr
inositol to the shoot. The experimental work is presented in the form
of three individual research papers, one of which has been published
while the other two are in manuscript form. The literature review gives
a brief history of the work leading up to this study and more specific
references can be found in the introduction sections of the research

papers.

LITERATURE REVIEW

The classic concept of auxin relationships in a young seedling
involves movement of a seed auxin precursor to the tip of the shoot or
root, conversion of the auxin precursor to an active auxin and then
transport of the active auxin to the growing region where hormonal
control can be exerted (Went and Thimann, 1937, p 65). The early work
leading to this concept has been thoroughly reviewed (cf. Boysen-Jensen,
1936, Went and Thimann, 1937, Went, 1951, Haagen-Smit, 1951) and more
recently reexamined (Ehmann, 1973). The chemical form of the auxin
precursor is however still a matter of debate.

The term auxin has been defined by a committee of the American
Society of Plant Physiologists, which in part reads: "a generic term
for compounds characterized by their capacity to induce elongation in
shoot cells" (Tukey et al., 1954, Larsen, 1954). In this review auxin
will be used to refer to preparation and compounds which have been
tested for by this biological activity, regardless of whether its
chemical structure is known or not.

Skoog (1937), in developing a more sensitive Avgng_curvature
bioassay for auxin, first demonstrated the long held assumption that the
seed is the source of precursors for auxin synthesized in the shoot tip
of seedlings. By de-seeding the plant he showed that the source of
auxin for the coleoptile tip is removed; the regeneration of the
physiological tip is prevented in decapitated coleoptiles, and thus more

4

5
sensitive test plants are obtained. He was also able to collect a sub-
stance from the decapitated coleoptile stump into agar blocks which gave
no curvatures with his test method in the first 5 to 6 hours (as would
be expected if the substance were and active auxin) but gave curvature
10 to 20 hours after application presumably by its conversion to an
active auxin. Skoog (1937) also demonstrated that tryptophan, which

Thimann (1935) had shown to be a precursor of IAA in Rhizopus suinus,

 

and tryptamine would behave in exactly the same manner in the de-seeded
Aggga_test as the precursor collected from the coleoptile stumps. Went
and Thimann (1937, p 65) proposed that the seed auxin precursor was an
auxin ester. This suggestion was, in part, based on the previous work
of Kogl et al. (1934) who obtained considerable yields of auxin after
the saponification of various oils prepared from seeds.

Van Overbeek (1941) compared the amounts of auxin obtained from
1 mm coleoptile tips of gea_by exhaustive diffusion and by exhaustive
extraction. Assuming that auxin obtained by exhaustive extraction was
active auxin and that obtained by exhaustive diffusion to be active
auxin present in the tip at excision plus active auxin derived from
precursor, Van Overbeek estimated that auxin precursor accounts for 92%
of the potential auxin in the ;gg_coleoptile tip.

Haagen-Smit et al. in 1941 were the first to isolate IAA from a
higher plant. Indole-3-acetic acid was isolated from alkaline hydro-
lyzed cornmeal and identified by its color reaction with ferric chloride
and lack of melting point depression when mixed with synthetic IAA.
Since the amount of IAA isolated after alkaline hydrolysis was much
greater than the amount of auxin activity extracted without alkaline

hydrolysis they proposed that most of the IAA must be in a bound form

(presumably an ester).

Until this time IAA had been assumed to occur only in lower
plants. The endogenous auxin active in higher plants was thought to be
either "auxin A" or "auxin B", chemically related compounds which had
been isolated from urine, malt and corngerm oil (kogl et al., 1933,
1934). The fact that no one else was ever able to repeat the isolation
of these two compounds did not prevent a long debate (which lasted into
the 1950's) as to which of the three compounds were the active auxins
in higher plants (Haagen-Smit, 1951, Went 1951). Avery et al. (1941)
demonstrated that the auxin of maize endosperm was very stable to heat
and strong alkali (5N NaOH) but was completely destroyed by autoclaving
in strong acid (2.5N HCl). They concluded that because of these
properties the auxin could not be identical with auxins A or B. These
properties are, however, consistent with the auxin being IAA. It is
currently thought that IAA is the principle active auxin in plants
(Bandurski and Bonner, 1952, Jacobs, 1979), and that auxin A and auxin B
were artifacts.

Berger and Avery (1944a) confirmed the earlier observation of
Haagen-Smit et al. (1941) of the presence of IAA in cornmeal by isolat-
ing an auxin precursor which, after alkaline hydrolysis, yielded IAA.
Berger and Avery (1944b) further characterized this precursor, showing
that it released IAA after mild alkaline hydrolysis (pH 9.5 for 5 min),
as would be expected for and ester-linked complex. The precursor was
very resistant to conversion to IAA by a variety of proteolytic enzymes
and had a low nitrogen content (4.7%) suggesting that the precursor was
not a protein. They also tested.zein, whole corn gluten, tryptophan and

three tryptophan containing proteins all of which yielded only extremely

7
small amounts of auxin activity as compared to the isolated precursor
when tested under the same conditions. They thus concluded that none
of theses substances was the precursor.

This led to twenty more years of attempts at characterizing the
bound auxins and auxin precursors of higher plants (cf. Bentley, 1958,
1961, Gordon, 1961, Cohen and Bandurski, 1982).

The first breakthrough was the demonstration by Andreae and Good
(1955) that exogenous IAA can be converted to IAA-aspartate by pea shoot
sections. This was the first chemical identification of an auxin con-
jugate formed jn_vjyg, Its Rf value and solubility properties suggest
that IAA-aspartate may account for a number af auxin active compounds
previously isolated but not characterized (Bentley, 1961).

Shantz and Steward (1957, Steward and Shanz, 1959) reported the
isolation of a chromatographically pure endogenous substance from im-
mature corn kernels which, upon hydrolysis, yielded IAA and arabinose.
This compound was later thought (Cohen, 1979) to be identical with and
IAA-m o-inositol-arabinoside isolated from mature corn kernels an
characterized by Labarca et al. (1965).

From 1961 to 1974 a series of papers from Bandurski's laboratory
reported work which identified and determined the concentration of all
of the IAA-containing compounds of mature Zea m§y§_kernels present in

1 or greater. This work has been thoroughly reviewed

amounts of long-kg'
(Ehmann, 1973, Cohen, 1979, Cohen and Bandurski, 1982) and the results
are summarized as follows: IAA accounts for approximately 1% of the
total IAA-containing compounds of the kernel; IAA-m o-inositols, 15%;
IAA-mygfinositol-arabinosides, 23%; IAA-m o—inositol-galactoside, 8%;

and the 01-97 and TriegylAA-m o-inositols and the 2-9:. 4:9:. and 6-9:

8
IAA-D-glucopyranoses all together accounting for less than 1% of the
total IAA; and IAA-glucans, where the glucan is a 3-1-4-cellulosic-
glucan with 7 to 50 glucose units per IAA, accounting for the remaining
53% of the total IAA. The total amount of all of the IAA-containing
compounds in the dry corn kernels used was approximately 76 mg-kg'l.

Epstein et al. (1980) determined the pool sizes and metabolic
turnover rates of a number of the indolylic compounds in the kernel of
the germinating corn seedling. The isotope dilution technique was used
to measure the amounts of tryptophan, tryptamine, IAA and IAA-myo-
inositol present in the kernel at zero time. Continued dilution of the
applied labeled compound, as a function of incubation time, was then
used to estimate turnover. Knowing the pool size and rate of turnover,
it is possible to estimate the specific activity of applied radiolabeled
compounds in the kernel. With this Epstein et al. (1980) were able to
calculate from data of Hall and Bandurdki (1978) on the amount of
radioactivity in IAA in the corn shoot derived from either radiolabeled
IAA or radiolabeled tryptophan applied to the endosperm, that neither
IAA or tryptophan in the endosperm supplied sufficient IAA to the shoot
to supply its estimated auxin needs. Thus, other compounds in the seed
must be the source of the IAA for the growing shoot.

IAA-mygyinositol esters constitute 30% of the low molecular
weight IAA derivatives in the corn kernel and thus are attractive
candidates as seed auxin precursors of the shoot. Nowacki and Bandurski
(1980) studied the translocation of radiolabeled IAA-mygrinositol from
the endosperm to the shoot of 4 day old dark-grown corn seedlings. They
found that the ratio of radiolabeled free IAA to radiolabeled ester IAA

in the shoot, after applying radiolabeled IAA-m o-inositol to the

9
endosperm, was 7:93 which is identical to the ratio found for endogenous
free to ester IAA (Bandurski and Schulze, 1974). Thus the translocated
radiolabeled IAA-mygfinositol can be hydrolyzed in the shoot to release
IAA and it appears to be equilibrating, at least to the extent of its
free to ester ratio, with the endogenous IAA compounds in the shoot.
Utilizing the data on pool size and turnover rates of IAA-mygfinositol
in corn kernels (Epstein et al.,1980) they calculated that IAA-m o-
inositol in the endosperm was traslocated to the shoot at a rate of
6.3 pmole into one shoot in one hour (Nowacki and Bandurski, 1980).
This amount is sufficient to supply the estimated 5 to 9 pmole of IAA
needed per shoot per hour (Nowacki and Bandurski, 1980).

EXPERIMENTAL I

Journal of Labelled compounds 5 Radiopharmaceuticals - V01, XIX, N0. 1 121

ENZYMATIC SYNTHESIS OF 5-3N-INDOLE-3-ACETIC ACID
AND 5-3H-INDOLE-3-ACETYL-Mlg-INOSITOL
FROM 5-3H—L-TRYPTOPHAN

Lech Michalczuk and John R. Chisnell
Department of Botany and Plant Pathology
Michigan State University
East Lansing, Michigan 48824
U.S.A.

SUMMARY

Labeled l-tryptophan is converted to indole-S-acetamide
and then to indole-3-acetic acid by enzymes from Pseudomonas
savastanoi. Labeled indole-S-acetic acid can be converted to
indole-3-acetyl-I:Q:§-D-glucose and to indole-B-acetyl-mygf
inositol by enzymes from kernels of Le; m_ay_s sweet corn.

Key Words: l-tryptophan, indole-S-acetamide, indole-3-acetic acid, indole-3-
acetyl-legrgrD-glucose, indole-3-acetyl-myg-inositol.

INTRODUCTION
High specific activity 3H-indole-3-acetic acid (IAA) is comuercially
available only by custom synthesis. However, 5-3H-l-tryptophan can be convert-
ed to 5-3H-indole-3-acetic acid in 70% yield, and at one-tenth the cost, by

enzymes from Pseudomonas savastanoi by the following reaction sequence (1,2):

0362-4803/82/010121-08501.00 Received September 22, 1980
©1982 by John Wiley 6 Sons, Ltd. Revised May 4, 1981

ll)

11

122 L. Michalczuk and J.R. Chisnell

 

a) l-tryptophan + 02 44: indole-3-acetamide + C02 + H20

.2) indole-S-acetamide + 2H20 --——-—>- indole-3-acetic acid + NH40H
Labeled IAA may then be converted to indole-3-acetyl—myg-inositol by enzymes
from kernels Of gga_mgy§_by the reaction sequence (3):

g) indole-3-acetic acid + UDPG .._____.. indole-3-acetyl-1-Q:§-D-glucose

g) indole-S-acetyl-1-Q¢_fD-glucose + Exp-inositol --- indole-3-acetyl-

gig-inositol + glucose
The enzyme catalyzing reaction a has been characterized as tryptOphan Z-mono-
oxygenase (EC 1.13.12.3) (1, 4); the enzymes catalyzing reactions b, g and d
have been characterized only as to the products formed (1, 3).

A chemical synthesis of 14C-indole-B-acetyl-myg-inositol employing 14C-
IAA-imidazole as the acylation reagent and mygeinositol as the alcohol acceptor
has been published (5) as has an enzymatic synthesis Of 14C-IAA using a similar
Pseudomonas savastanoi enzyme preparation (2). However, the enzymatic
syntheses here described are adapted to a high specific activity, microscale
synthesis of 3H-IAA-m O-inositol and 3H-IAA. 3H-IAA has been used to study
transport (6) and metabolism (cf. 7) of IAA in plants and in a radioinmunoassay
for IAA (8). IAA-myg-inositol is a major ester Of IAA in leg (9), and the
[labeled compound has been used to study turnover of the ester (10) and its

transport (II).

EXPERIMENTAL

Materials:

5-3H-l-tryptophan, specific activity 29 Ci/mmol (210%), was obtained from
Research Products International Corp. (a product Of CEA, France) and used with-
out isotopic dilution or prior purification. It was 75% radiochemically pure
as determined on thin layer chromatograms (Silica Gel 60 tlc plates, E. Merck,
Darmstadt) developed with Solvent A (methylethylketone-ethylacetate—ethanol-
water [3:5:1:1]). [2-ring-14CJindole-3-butyric acid (14C-IBA), specific acti-

vity 508 uCi/mmole, was synthesized by Dr. J.D. Cohen (12). High performance

12

3 . .
Enzymatic Synthesis of 5- H—IndoZe-3-Acettc Aczd

liquid chromatography (HPLC) was performed on a Whatman PXS 10/25 ODS reverse
phase column (25 cm x 0.46 cm) with a Whatman CO:PELL ODS precolumn (7 cm x 0.2
on) (Whatman Inc., Clifton, NJ). Tetrahydrofuran was redistilled over
potassium metal under an N2 atmosphere inmediately prior to use. Gas-liquid
chromatography was performed on a Varian 2740 gas chromatograph equipped with
both a Varian TSD nitrogen specific thermionic detector and an FID with N2 as
carrier gas. Two 6 ft x 2 mm ID glass columns packed with 3% 0V17 on 100/120
Gas Chrom 0 (Applied Science, State College, PA) were used, one with the
nitrogen specific detector for* determination Of the relative quantities of
compound and one in conjunction with an FID for peak collection to determine
radioactivity. Scintillation counting was performed with a Beckman LS7000
scintillation counter. SP Sephadex C-25 was from Pharmacia Inc., Piscataway,
NJ; UDPG, indole-3-acetic acid and m O-inositol were products of Signa Chemical
CO., St. Louis, MO, and used without purification.

Engyme preparation:

Pseudomonas savastanoi was grown as described by Kosuge et al (1),
harvested by centrifugation and stored as a cell paste in liquid nitrogen. All
enzyme preparative steps were performed at 0-3°. Approximately 10 g of frozen
cells were thawed and suspended in 50 ml Of glass distilled water, resedimen-
ted, and 'this procedure repeated three times. The pellet from the third
centrifugation was suspended in 50 ml Of 0.01 M potassium phosphate buffer, pH
7.6, containing 5 TIM mercaptoethanol. The cell suspension was sonically
disrupted (Branson Model 125, Branson Instruments, Inc., Danbury, Conn.) for
five 30 sec pulses with 30 sec intervals between each pulse, and the resultant
homogenate was centrifuged at 20,000 x g for 15 min. To the supernatant fluid
(50 ml total volume), 15.6 g of solid (NH4)2504 (enzyme grade, Mann Research
Lab., Inc., New York, NY) was added, the resultant precipitate sedimented by
centrifugation and then dissolved in 5 ml of 0.05 M Tris-HCl, pH 7.6, contain-
ing 5 mM mercaptoethanol. This solution was desalted on a 30 ml bed voluue

Sephadex 6-50 column eluted with the buffer used to dissolve the Uhh)2804

123

13

124 L. Michalczuk and J.R. Chisnell

pellet. The desalted enzyme (10 inl total volume) was divided into 1 ml
aliquots and stored at -70° for further use. Under these conditions both
decarboxylating and hydroxylating enzymes were stable for at least 2 weeks.

An enzyme preparation from Zea, catalyzing reactions g and g, was prepared
by the method Of Kopcewicz et al (13). Stage I enzymes (the 10,000 x 9 super-
natant of the crude homogenate) were made 85% saturated with respect to
(NH4)2SO4 by the addition of the solid salt. The precipitated proteins were
suspended in 0.01 M Tris-HCl, pH 7.1, buffer and desalted on a Sephadex G-50
column to yield Stage II enzymes (3). This preparation, in 5.0 ml aliquots
stored at -70°, was stable for at least 10 weeks.

Product Characterization:

Indole-3-acetic acid: The time course for the conversion of 5-3H-l-tryto-
phan to labeled indole-3-acetamide and IAA was followed by thin layer chromato-
graphy of aliquots of the enzyme reaction mixture. With Solvent A on Silica
Gel 60, authentic l-tryptophan, indole-3-acetamide and IAA are at Rf 0.13, 0.75
and 0.87 respectively. The putative IAA was at Rf 0.87. The Rf of the puta-
tive IAA iii another solvent system, chloroform-methanol-water (85:14zl), on
Silica Gel 60 was also identical to that Of authentic IAA (Rf 0.23). The GC
retention time Of the methyl ester, prepared using diazomethane (14), was
identical to that Of the "ethyl ester of authentic IAA. The identity of IAA
was further established by its conversion to IAAgmyQ-inositol.

The radiochemical purity of 3H-IAA was determined by thin layer chroma-
tography in two solvent systems (Solvent A and chloroform-methanol-water
[85:14:11) on Silica Gel 60.

The specific activity of the 3H-IAA was determined by a modification of
the method Of Cohen and Schulze (12). Approximately 2 nmole 14C-IBA was added
to 1 nmole biosynthetic 3H-IAA. Methylation Of the mixture and gas chromato-
graphy using the nitrogen specific detector (12) showed the mole ratio of IBA
to IAA to be 0.78. Chromatography of a second aliquot of the mixed methyl

esters with collection Of the radioactivity at the retention times of IAA and

14

. . 3
Enzymatic synthesis 0f 5- H-IndoZe-3-Acetic Acid

and IBA showed the ratio of the radioactivities of IAA to IBA to be 70,800. As
can be deduced from the equations of Cohen and Schulze (12), the specific acti-

vity of the IAA in the mixture can be calculated by the following equation:

(dpm IAA)(IIDIES IBA
dun IBA moles IAA

)(specific activity IBA) = specific activity IAA
The specific activity of the 3H-IAA was determined to be 28 Ci/mmole.

Indole-3-acetyl-m o-inositol: The putative mixed isomeric IAA-mygeinosi-
tols (15) were eluted frmn a 0.9 )t 17 cm HPLC column Of sulfonated styrene-
divinylbenzene copolymer (Beckman PA-28) (5,16) with retention times identical
to those of authentic IAA-m o-inositols. They had the same Rf values on thin
layer chromatograms (Silica Gel 60 developed in Solvent A) as did authentic IAA-
myg-inositols (Rfs 0.28 and 0.32). Upon ammonolysis in 15% NH4OH for 45 min at
45°, the methyl ester prepared from the sample had a GC retention time identical
to that of the methyl ester Of authentic IAA. Ammonolysis Of IAA esters yields
the free acid and the amide. The amide was not volatile in the GC system used
and was not detected. In a previous paper (3), GC-MS analysis of the trimethyl-
silyl derivatives of the reaction products of leg enzyme Stage II and unlabeled
authentic IAA showed mass spectral fragnentation patterns identical to those of
authentic hexakis trimethylsilyl-IAA-myg-inositol.

The radiochemical purity of 3H-IAA-myg-inositol was determined by thin lay-
er chromatography on Silica Gel 60 developed in Solvent A.

The specific activity of the 3H-IAA-mygeinositol was determined after puri-
fication to 94% radiochemical purity by first chromatographing it over an
SP-sephadex column (1 ml bed volume) eluted with Z-propanOl-Hzo (1:1) and then
chromatographing it on a reverse phase HPLC column eluted with tetrahydrofuran-
H20 (18:82). The ester was ammonolyzed to free IAA, and the procedure described
above was followed exactly. Approximately 3 nmole 14C-IBA was added to 0.5
nmole 3H-IAA from ammonolyzed biosynthetic 3H-IAA-myg-inositol. The mole ratio
of IBA to IAA was 2.13 and the ratio of radioactivity in IAA to IBA was 24,500.

The specific activity of 3H-IAA-myg-inositol was calculated to be 27 Ci/mmole.

125

15

126' L. Michalczuk and J.R. Chisnell

Enzymatic Conversions:

5-3H-indole-3-acetic acid: The 5-3H-l-tryptophan (1.5 mCi, 0.05 umol) in
1.5 ml of water was incubated with 1.0 ml of the Pseudomonas enzyme preparation
in 0.05 M TrisHCl, pH 7.6, buffer for 2 hr at 37°. The reaction proceeded to
90% completion as determined by thin layer chromatography and was terminated by
the addition of an equal volume of 2-propanol. Denatured proteins were removed
by centrifugation and the supernatant fluid applied to a 0.6 x 20 cm DEAE-
Sephadex-acetate column. The column was developed with 300 ml of a linear
gradient of 1:1 2-propanOl-water to 1:1 2-propanol-0.04 M potassium phosphate
(pH 3.5). Elution was at 0.04 M phosphate (140 to 154 ml). Tubes containing
more than 5% of the total eluted radioactivity were combined. The IAA was
extracted into ether (4 x 14 ml), the ether dried over Na2504 and evaporated to
near dryness, and the IAA taken up in 5 ml of acetonitrile. The yield was 68%,
the radiochemical purity was 91%, and the specific activity was 28 Ci/mmole.

5-3H-indole-3-acetyl-m O-inositol: The 5-3H-l-tryptophan (1.5 mCi, 0.05
umol) in 1.5 ml water was incubated with 1.0 nfl of the Pseudomonas enzyme
preparation and 1.5 ml of the Egg enzyme preparation Stage 11, together with 5
pmol of UDPG and 5 umol of Eye-inositol in a total volume Of 4 ml. The reac-
tion was stopped by adding an equal volume of 2-propanol; the product was freed
of anionic materials and purified on the PA-28 HPLC column developed with 1:1
2-propanOl-water (3). The resultant product was stored in 1:1 2-propanol-water
at -20° in sealed ampules. The yield was 40% based on l-tryptOphan, the
radiochemical purity was 70%, and the specific activity was 27 Ci/mmole.

Incubation of the tryptophan with both enzyme preparations simultaneously
provided better yields Of IAA-myg-inositol than did converting tryptophan to
IAA and later esterifying the IAA to m O-inositol.

16

. . 3 . .
Enzymattc Syntheses of 5- H-IndoZe-R-Acettc Actd

DISCUSSION

Our results indicate that high specific activity 3H-tryptophan can be
enzymatically converted to 3H-IAA and 3H-IAA-myg-inositol with little or no
dilution of the specific activity. The determined specific activities of the
products are within 10% of that Of the manufacturer's stated specific activity
of 29 Ci/mmole for the 3H-l-tryptophan substrate. The manufacturer‘s accuracy
in the specific activity determination was 110%.

The modification of the specific activity determination method of Cohen
and Schulze used here is a simplification Of their calculations. This simpli-
fication eliminates the requirement for knowing the amount of internal standard
added to the sample (in this case 14C-IBA); only the specific activity need be
known. It also eliminates the requirement that the isotopically labeled inter-
nal standard be chemically related, and requires only that the standard contain
nitrogen and that the relative nitrogen specific detector response to the two
compounds be determined. Lastly, by reducing the number of calculations the

accuracy of the method is improved.

ACKNOWLEDGEMENTS

 

We thank Professor T. Kosuge for cultures Of Pseudomonas savastanoi, and
Dr. J. Cohen for suggesting this enzymatic synthesis and providing the l4C-IBA.
We also thank Miss H. La Maine for her expert assistance in manuscript prepara-
tion. This work was supported by the Metabolic Biology Section of the National
Science Foundation grants PCM 76-12356 and PCM 79-04637 to Professor R. S.
Bandurski and is journal article 9643 from the Michigan Agricultural Experiment

Station.

12.7

128

17

L. Michalczuk and J.R. Chisnell

REFERENCES
Kosuge T., Heskett M.G. and Wilson E.E. - J. Biol. Chem. 111: 3738 (1966)
Hutzinger 0. and Kosuge T. - In "Biochemistry and Physiology of Plant
Growth Substances", Wightman, F. and Setterfield, G. editors, The Runge
Press LTD., Ottawa. Canada, (1968)
Michalczuk L. and Bandurski R.S. - Biochem. Biophys. Res. Comm. 91: 588
(1980)
Comai, L. and Kosuge, T - J. Bacteriol. 111: 950 (1980)
Nowacki J., Cohen J.D. and Bandurski R.S. - J. Labelled Compd. RadiOpharm.
15: 325 (1978)
Gardner 6., Shaw S. and Wilkins M.B. - Planta 111: 237 (1974)
Davies P.J. - Physiol. Plant. 18: 95 (1973)
Pengelly W. and Meins F. - Planta 116: 173 (1977)
Ueda M. and Bandurski R.S. - Phytochem. 11: 243 (1974)
Epstein E., Cohen J.0. and Bandurski R.S. - Plant Physiol. 65; 415 (1980)
Nowacki J. and Bandurski R.S. - Plant Physiol. 65: 422 (1980)
Cohen, J.D. and Schulze, A. - Anal. Biochem. - In Press.
Kopcewicz J., Ehmann A. and Bandurski R.S. - Plant Physiol. 51: 846 (1974)
Schlenk, H. and Gellerman, J.L. - Anal. Chem. 3;: 1412 (1960)
Ehmann A. and Bandurski R.S. - Carbohydr. Res. 36: 1 (1974)
Cohen J.D. and Bandurski R.S. - Plant Physiol. 59(5): 10 (1977)

18
EXPERIMENTAL II

MlgleOSITOL ESTERS OF INDOLE-3-ACETIC ACID ARE

ENDOGENOUS COMPONENTS OF 1§A_MAYS L. SHOOT TISSUE1

John R. Chisnen2
Department of Botany and Plant Pathology
Michigan State University
East Lansing, Michigan 48821-1312

Manuscript received date:

19
Footnotes

1Supported by grants from the Metabolic Biology Section of the National
Science Foundation, PCM 8204017, 0RD 30668, and the Life Science Section
of the National Aeronautics and Space Administration, NAGW 97, 0RD 25796
to Professor R.S. Bandurski. This is Journal Article NO. 10863 fron the

Michigan Agricultural Experiment Station.

2Present address: Department of Microbiology, North Carolina State

University, Raleigh, NC 27650.

3Abbreviations: IAInos, Indole-3-acetyl-mygrinositol; FID, flame
ionization detector; TSD, thermionic specific detector; PVPP,
polyvinylpolypyrrolidone; [14CJIBA, [2-ring-14CJindole-3-butyric
661d; [14CJIAAsp, [2-acetyl-14CJindOle-3-acetyl-L-aspartate.

20
Abstract

Indole-3-acetyl-mygeinositol esters have been demonstrated to be
endogenous components of etiolated 1gg_m§y§_shoots tissue. This was
accomplished by comparison of the putative compounds with authentic,
synthetic esters. The properties compared were liquid and gas-liquid
chromatographic retention times and the 70 eV mass spectral
fragmentation pattern Of the pentaacetyl derivative. The amount of
indole-3-acetylemygeinositol esters in the shoots was determined to be 74
nmol kg fresh weight as measured by isotope dilution, accounting for
19% of the ester indole-3-acetic acid of the shoot. This work is the
first characterization Of an ester conjugate of indole-3-acetic acid from
vegetative shoot tissue using multiple chromatographic properties and
mass spectral identification. It is of interest that the kernel and the
seedling shoot both contain indole-3-acetyl-mygeinositol esters, and that
these esters comprise approximately the same percentage of the total

ester content of the kernel and Of the shoot.

21
Introduction

Early work on Axegg by Skoog (23) indicated that the seed was the
source of an auxin precursor transported to young dark grown shoots.
Went and Thimann (25) suggested that this auxin precursor might be an
ester. Previous work in this laboratory has shown that the IAA ester,
indole-3-acetylHmyg-inositol (IAInos)3, is a major component of the 123
gays endosperm (12,24), and that ester conjugates of IAA constitute up to
80% Of the IAA in the shoots Of young dark grown 1e; (3). The presence
of alkali-labile auxin complexes in gee xylem sap was demonstrated by
Sheldrake (20), who suggested that the proposed seed auxin precursor was
an ester transported in the xylem. Later, Nowacki and Bandurski (16)
demonstrated that exogenous radiolabeled IAInos was transported from the
endosperm tO the young dark grown shoot and could be recovered from the
shoot both as intact IAInos and as free IAA. However, until now IAInos
has not been shown to be an endogenous component of the shoot tissue. In
this report we demonstrate that IAInos is an endogenous component of 133
shoot tissue and provide an estimate of the amount present in the shoot
tissue. This report corrects a previous abstract Of these studies (4)
which contained erroneously high calculations of the amounts of IAInos
and free + ester IAA in the shoot tissue. This report adds to the
increasing body of literature pointing to the importance of IAA

conjugates in vegetative tissue (1,8,13).

22
Materials and Equipment

Radioactivity was measured with a Beckman LS 7000 scintillation
counter. HPLC was performed with a Varian 5060 liquid chromatograph
equipped with a Varian UV-5 280 nm detector. GLC was on a Varian 2740
gas chromatograph equipped with an F10 and a TSD adjusted for organic
nitrogen detection. GC-MS was performed with a Hewlett Packard 5985a
quadrupole mass spectrometer.

Materials were from the following sourceszinsoluble PVPP, Dowex
50W-X2 200-400 mesh and DEAE sephadex:Sigma; sulfonated styrene-divinyl
benzene PA-28 resinzBeckman; 3% OV-17 on 100/120 Gas Chrom QzApplied
Science; Partisil 10, Partisil 10-ODS, CO:Pell ODS, and HC Pellosil HPLC
column packing materials: Whatman; Zegmmgys cv. Stowell's Evergreen
Hybrid Sweet CornzBurpee Seed CO., 4-dimethylaminopyridine:Aldrich.
[14CJIDA (508 uCi/mmol), [14CJIAAsp (54.5 uCi/umol) and IAInos were
synthesized (5,9,17) and kindly provided by J.D. Cohen (USDA,
Beltsville). Indole-3-acetyl-DL-aspartate was synthesized by the method
of Mollan £1.31. (15) and kindly provided by R.P. Hangarter (Michigan
State University). [5-3HJIAInos (two batches, 29 Ci/mmol and 27 Ci/mmol)
was synthesized (14) from L-[5-3HJtryptOphan (29 Ci/mmol and 27 Ci/mmol,
respectively) obtained from Research Products International. A
[1-acetyl-14CJIAA (59 mCi/mmol) was obtained from Amersham and
[2-acetyl-14CJIAA (42.2 mCi/mmol) was obtained from New England Nuclear.

Pyridine and acetic anhydride, for acetylations, were distilled and
stored in a desiccator over anhydrous CaSO4. Tetrahydrofuran was

freshly redistilled over potassium metal in a nitrogen atmosphere.

23
Methods and results

Plant material. Corn kernels were surface sterilized in 1% NaOCl
for 10 min, soaked in running tap water (21°C) for 24 h and then sown in
flats Of vermiculite and watered with distilled water. Growth was for 5
d in darkness at 25°C. A phototropically inactive green light was used
during manipulations for growth and harvest.

Isolation Of IAInos. Whole corn shoots were cut approximately one
to two cm above the seed and drOpped into liquid nitrogen. Prior to
grinding, the liquid N2 was evaporated and the shoots weighed to
provide a fresh weight estimate. The shoots were ground in a blender
with sufficient acetone to give a tissuezacetone ratio of 3:7 (w/v). TO
the acetone was added a known amount Of [3H]IAInos (approximately 4
uCi-kg-l) and [14CJIAA (approximately 2 uCi-kg-l) and sufficient
2-mercaptoethanol to make the final concentration 50 mM. The tissue was
extracted for 24 h at 22°C, and the mixture filtered through Whatman No.
1 paper on a Buchner funnel. The retained acetone insoluble material was
dried, weighed, and used as an estimate Of the dry weight Of the tissue.
Aliquots of the filtrate were taken for quantitative determination of
free and free + ester IAA. The acetone was distilled from the filtrate
i_n_ y_a_c_u_g and the remaining aqueous solution (pH 4) was partitioned twice
with CHCl3 and twice with diethyl ether. After reducing the pH of the
solution to 3.5 with H3P04 it was again partitioned. These partitionings
removed impurities and extracted 97% of the 14C. Residual solvent was
removed and the remaining solution reduced in volume ig_ygggg_to 130 ml.
The sample was then placed on a PVPP column (4.1 cm x 22 cm) and eluted
with 50 mM NaAcetate, pH 3.5. containing 10 mM 2-mercaptoethanol. 3H

containing fractions eluting between 250 ml and 700 ml were pooled, and

24
the volume reduced lg vacuo to 90 ml. The sample was next placed on a

short Dowex column (SOW-XZ, Na+ form, 9.6 cm x 4 cm in a fritted disc
Buchner funnel) and eluted with 50% (v/v) aqueous 2-propanol containing
10 mM 2-mercaptoethanol, using suction. Fractions Of 50 to 200 ml were
collected with the 3H containing fractions eluting at 300 to 1200 ml.

The first 25% of the 3H fractions were tOO impure for use in the follow-
ing step. Therefore, these fractions were pooled and rechromatographed
on Dowex 50W-X2. The last three-fourths of the 3H fractions from the
second column were pooled with those from the first Dowex column. The
volume was reduced in vacuo to 3 ml and placed, in 3 batches, on a PA-28
column (0.9 x 17 cm) (7) and eluted with 50% (v/v) aqueous 2-propanol
containing 10 mM 2-mercaptoethanol at a flow rate of 11 ml-hr-l. During
concentration Of the Dowex fractions a precipitate formed which could be
removed by centrifugation without significant loss of 3M. This was

also the case during the concentration procedure preceeding all of the
following liquid chromatographic steps. IAInos containing fractions
(elution volume 20 to 40 ml) from the three PA-28 runs were pooled, the
volume reduced lg 13219, loaded, in 3 batches, on a Partisil 10 DDS 013
reverse phase column (0.46 x 25 cm) with a CO:Pell ODS precolumn (0.21 x
7 cm) and eluted with 5% (v/v) aqueous ethanol at a flow rate of 1
ml-min-l. The mixed isomeric IAInos (10) elute in 3 peaks with maxima at
9, 12, and 15 ml. The 3H containing fractions eluting at the IAInos
elution volumes from the three Partisil 10 ODS runs were pooled and
evaporated ih,ygggg_to about 30 ul Of a viscous brown oily residue. This
was dissolved in ethyl acetatezacetonitrilezethanol:HZO (5:3:3zl, v/v),
volume 300 pl, and placed on a Partisil 10 normal phase column (0.46 x 25

cm) with an HC Pellosil precolumn (0.21 x 7 cm) and eluted with ethyl

25
acetatezacetonitrile: ethanol:H20 (10:3:3:1, v/v) at a flow rate of 0.5

ml-min-l. The IAInos elutes as a single peak from 4 to 9 ml with a
maximum at 6 ml. The IAInos fractions were pooled and evaporated to
dryness. Then, using an Nz-flushed dry box, the residue was dissolved

in 50 ul of pyridine containing 0.5% (w/v) 4-dimethylaminopyridine
followed by the addition of 50 ul of acetic anhydride. The mixture was
reacted for 10 min at 22°C to permit acetylation, then evaporated under a
stream of N2 to a thin brown oily residue. This was dissolved in 28%
(v/v) aqueous ethanol to a final volume of 200 pl. The sample was next
placed on a Partisil 10 ODS column and eluted with 28% (v/v) aqueous
ethanol at a flow rate of 2 ml-min-l. The acetylated IAInos elute in two
peaks with maxima at 38 and 42 ml. Approximately one half of the 3H
recovered from this column eluted at volumes other than those for IAInos
and was presumed to be decomposition products or underivatized material.

Recovery of [3H]IAInos at this step was approximately 20%.

Qualitative analysis

GLC. Acetylated IAInos fractions from the Partisil 10 ODS column
were pooled, evaporated to dryness ig_ygggg, and the sample dissolved in
tetrahydrofuran. GLC, using the TSD, was isothermally at 300°C on 3% CV
17 (2 mm x 1.8 m) using N2 as the carrier gas. The acetylated derivative
Of authentic mixed isomeric IAInos elutes as four peaks in this system
(Figure 1). In the acetylated plant sample two major peaks (peaks 1 and
3) have been detected (Figure 1) with the T50.

It was important to determine whether the recoveries of all the
isomers of IAInos by our purification procedure were equivalent. In

Experiment 2 the amounts of 3H, eluting at the retention times for the

26
four IAInos GLC peaks, was determined for the [3H]IAInos in both the pure
authentic material, and after it had been reisolated from the plant
sample. The [3H]IAInos was diluted approximately 1:300 with IAInos,
acetylated, the acetylating reagents evaporated, and the sample dissolved
in tetrahydrofuran. Four hundred and sixty thousand dpm (approximately 2
nmol) were injected onto an 0V 17 column, identical to the one described
above, but connected to an FID with the detector flame extinguished. The
radioactivity eluting between 0.25 to 18 min was collected and measured.
The results are presented in the bar graph Of Figure 2A, below a TSD
tracing of an injection of 1 nmole from the same sample. Recovery of the
injected 3H was 43%. The relative amounts of 3H eluting at the elution
times for the four acetylated IAInos peaks was 11:2:79:9 (P1:P2:P3:P4).
Forty eight thousand dpm (approximately 0.5 nmole) Of the acetylated
putative IAInos reisolated from the plant sample was injected as above
and the radioactivity eluting between 0.25 to 32 minutes was collected
and measured. The results are presented in the bar graph of Figure 2B,
below an FID tracing of an injection of 7 nmoles of acetylated authentic
IAInos. Recovery of injected 3H was 36%. The relative amounts of 3
eluting at the elution times for the four acetylated IAInos peaks was
11:1:83z4 (P1:P2:P3:P4). The similarity in the ratios of the 3H under
the four peaks between the authentic [3H]IAInos and the [3H]IAInos as
recovered from the plant sample suggests that the isomers eluting in
peaks 1, 3, and 4 were equally recovered. The amount Of 3H under peak
2 in the plant sample was insufficient to rule out the possibility that

the 3H collected at that retention time represented tailing from peak 1.

27
GC-MS. In Experiment 2 sufficient material was recovered to permit

GC-MS analysis. Analyses were at a 70 eV ionizing potential and
chromatography was on 3% CV 17 (2 mm x 61 cm), temperature programmed at
200°C for 2 min followed by a temperature increase Of 20°C-min-1 to 300°C
with He carrier gas at a flow rate of 26 ml-min-l. The retention times
for peaks 1, 3, and 4 were 7.9, 9.0, and 9.4 min, respectively. Peak 2
was not observed. Table I lists the principal ions in the spectra Of
authentic and putative plant IAInos acetyl esters of peaks 1, 3, and 4.
Figure 3 shows the spectra of the authentic and isolated pentaacetyl

esters Of IAInos in peak 3.

Quantitative analysis

IAInos. The amount Of IAInos was determined by a modification of the
double-standard isotope dilution method of Cohen and Schulze (9,14). In
brief, the method utilizes coinjection into the GLC of a compound of known
specific activity (the second internal standard) with a compound of
unknown specific activity (the primary internal standard diluted by an
unknown amount of endogenous compound). The ratio of the radioactivities
collected at the elution times for the two compounds and the ratio of the
peak areas of the two compounds are determined. Then if the relative molar
response of the detector to the two compounds is known, the specific
activity of the unknown compound can be calculated. To determine the
specific activity of the [3H]IAInos reisolated from the plant sample the
following equation was used:

IAAsp peak area relative detector dpm IAInos

X specific activity
IAInos peak area

response IAInos/IAAsp X dpm IAAsp X Of the IAAsp

= the specific activity of the IAInos

28
[3H]IAInos was the primary internal standard, the second internal
standard was [14CJIAAsp methylated with ethereal diazomethane (19)
forming the bis-methyl derivative. The molar response Of the T50 to
acetylated IAInos relative to bis-methyl-IAAsp was determined to be 0.68.
The GLC system was as described for the qualitative analysis.
Representative chromatograms are presented in Figure 1. Knowing the
specific activity Of the reisolated IAInos it was possible to calculate
the amount in the plant sample using the isotope dilution equation

(18):

Y=[—c—g-1]x

where CO is the initial specific activity Of the added [3H]IAInos, C
is the specific activity Of the reisolated [3H]IAInos (determined as
indicated above), X is the molar amount of [3H]IAInos added to the
extract, and Y is the molar amount of IAInos in the plant sample. The
quantitative results for two experiments are summarized in Table 11.

Free and free + ester IAA. Aliquots for free and free + ester IAA
determination (approximately 5% and 3% by volume, respectively) were
taken from the acetone extracts used for the IAInos quantitative analysis
described above. Acetone was removed from the aliquots 1Q 32239. At
this step the samples for free + ester IAA determination were made 1N
with respect to NaOH by the addition Of an equal volume of 2N NaOH and
incubated for 1 hr at 22°C to hydrolyze the IAA esters (3). These
hydrolysis conditions would also hydrolyze any acyl anhydride or
thioester conjugated IAA, if present. The samples, both free and free +
ester, were then acidified to pH 3.5 with H3PO4, and extracted three

times with CHCl3. The CHCl3 layer was dried over anhydrous Na2504 and

29
then the CHCl3 was removed 15 vacuo. The residue was solubilized in 50%

(v/v) aqueous 2-propanol and placed on a DEAE-Sephadex (acetate form)
column (0.8 x 10 cm), washed with 20 ml Of 50% (v/v) aqueous 2-propanol
and eluted with a linear gradient of 50% (v/v) aqueous 2-propanol
containing from 0 to 5% (v/v) glacial acetic acid. Fractions containing
IAA (about 45 ml elution volume from beginning of gradient) were pooled
and dried ih_ygggg. The residue was solubilized in 30% (v/v) aqueous
ethanol containing 0.7% (v/v) acetic acid, loaded on a Partisil 10 ODS
column and eluted with the solubilizing solution at a flow rate of 1
ml-min-l. Fractions containing IAA (about 8 ml elution volume) were
pooled, dried lg 11919 and methylated with ethereal diazomethane. The
methylated product was dried under a stream Of N2 and dissolved in
tetrahydrofuran. The amount of IAA in the samples was determined by the
same method as used for IAAnos. The primary internal standard for the
free IAA quantitative determination was [14CJIAA. When the aliquot for
free + ester IAA determination was hydrolyzed, the [3H]IAInos tracer in
the sample was hydrolyzed, along with the plant ester IAA, yielding
[3H]IAA. Therefore, both [14CJIAA and [3H]IAInos (hydrolyzed to [3H]IAA)
served as parallel primary internal standards (requiring differential
counting for the two isotopes) in the determination of free + ester IAA.
[14CJIBA, methylated with ethereal diazomethane, was the second internal
standard. The GLC system was as described for the acetylated IAInos
qualitative analysis except that chromatography was isothermal at 200°C.
The results for two experiments are presented in Table II. The average
and relative amounts Of IAInos, free IAA, and free + ester IAA are

summarized in Table III.

30

Discussion

Endogenous IAInos was isolated from 5 day Old 1, mays shoot tissue.
It was identified by comparison of its physico-chemical properties with
authentic IAInos, and measured quantitatively by an isotope dilution
method. The mass spectra of the isolated compound were identical to
those of the authentic compound, both having a molecular ion at m/z 547.
Other ions, characteristic Of acetylated Eye-inositol, were at m/z 373
(M+-IAA), m/z 157, and m/z 103 (Ac20H+) (21). Ions characteristic
Of the indole moiety were at m/z 130 (the quinolinium ion ) and m/z 103
(the styryl ion) (26). The putative IAInos cochromatographed with
authentic [3H]IAInos in 5 liquid chromatographic systems. As the acetyl
derivative it cochromatographed with the authentic tritiated derivative
in the liquid chromatographic and GLC systems tested. Two of the 4
acetylated authentic IAInos peaks eluting in the GLC system were detected
with a ISO in the acetylated putative IAInos sample.

Since the isomers of IAInos elute as more than one peak in at least
one of the chromatographic purification steps, the possibility Of
preferential loss Of one or more of the isomers exists. Data suggesting
that preferential isomer loss was not a problem is presented in figure 2
and discussed in the Methods and Results section. However, acyl
migration, observed in inositol esters (2,22), has previously been noted
in IAInos esters (12). This might account for similar ratios of
[3H]IAInos in the 4 GLC peaks as long as acyl migration occurred after
the step in which a preferential loss might have occurred. If
preferential loss did occur the most likely isomers to be lost would have
been those eluting in peaks 2 or 4 in the GLC system. Loss of these

isomers was more apt tO be unnoticed since the labeled tracer contained

31
relatively little Of them and it was the label which was followed during

purification. If the shoot tissue contained relativly more Of these
isomers than that found in the labeled tracer and preferential loss Of
these isomers did occur, then the estimate for the amount in the tissue
may be low. If acyl migration occurred before preferential isomer loss
the isomers Of the endogenous IAInos and the isomers Of the added
[3H]IAInos would have come to the same equilibrium mixture and all of
the isomers would be equal in specific activity. Thus, the specific
activity Of any one isomer would reflect the amount of isotope dilution
encountered by the entire sample, and preferential loss of an isomer
would have had no effect on the accurate determination Of the amount Of
IAInos in the tissue.
IAInos accounts for about 15% of the free + ester IAA or about 19%

Of the ester conjugated IAA of the shoot. Thus, about 80% of the ester
IAA Of the shoot is some ester(s) other than IAInos. This ratio Of
IAInos to ester IAA is similar to that found in the kernel after 4 days
of germination (25%) (11). Other IAA esters in the shoot may be the same
as those found in the kernel, for example, IAInos-arabinosides,
IAInos-galactosides, di- and tri-O-IAInos, and IAA-glucosides (8). Due
tO the difficulty of isolating these types of neutral compounds, which
would only be present in very low amounts in the tissue, identification
and quantification Of the other IAA ester conjugates in the shoot awaits
the synthesis of suitable radiolabeled internal standards.

- The close agreement in the amount of free + ester IAA in the shoot
whether [14C]IAA or [3H]IAInos was used as the internal standard
validates the customary use (cf. 3) Of free IAA as the internal standard

in the determination of free + ester IAA. This confirms an earlier

32
validation noted by Cohen (6) when he compared values for total (free

+ ester + amide) IAA in seeds Of GLycine max L. determined using

 

[14CJIAA and [14CJIAAsp as internal standards.

33
Acknowledgements

I wish to thank Professor R.S. Bandurski for advice and support,
P.J. Hall for help with mass spectrometry, and M.D. La Maine for
assistance in manuscript preparation. Mass spectral data were Obtained
from the Michigan State University Mass Spectrometry Facility supported
by a grant (RR-00480) from the Biotechnology Resources Branch, Division

of Research Resources, NIH.

34

LITERATURE CITED

1.

7.

9.

Andersson B, G Sandberg 1982 Identification Of endogenous
N-(3-indoleacetyl)aspartic acid in scots pine (Pinus sylvestris L.)
by combined gas chromatography mass spectrometry, using
high-performance liquid chromatography for quantification. J
Chromatogr 238:151-156

Angyal SJ, GJH Melrose 1965 Cyclitols. Part XVIII. Acetyl migration:
Equilibrium between axial and equatorial acetates. J Chem Soc
1965:6496-6500

Bandurski RS, A Schulze 1977 The concentration of indole-3-acetic
acid and its derivatives in plants. Plant Physiol 60:211-213

Chisnell JR, RS Bandurski 1982 Isolation and characterization of
indole-3-yl-acetylemyg-inositol from vegetative tissue of 1gg_m§y_.
Plant Physiol 69:5-55

Cohen JD 1981 Synthesis of 14c-labeled indole-3-acetylaspartic
acid. J Labelled Comp Radiopharm 18:1393-1396

Cohen JD 1982 Identification and quantitative analysis Of
indole-S-acetyl-L-aspartate from seeds of Glycine max L. Plant

Physiol 70:749-753

 

Cohen JD, RS Bandurski 1977 The rapid separation and automated
analysis Of indole-3-acetic acid and its derivatives. Plant Physiol
59:S-10

Cohen JD, RS Bandurski 1982 Chemistry and physiology Of the bound
auxins. Ann Rev Plant Physiol 33:403-430

Cohen JD, A Schulze 1981 Double-standard isotope dilution assay 1.
Quantitative assay of indole-3-acetic acid. Anal Biochem

112:249-257

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

35

Ehmann A, RS Bandurski 1972 Purification of indole-3-acetic acid
Eye-inositol esters in polystyrene-divinylbenzene resins. J
Chromatogr 72:62-70

Epstein E, JD Cohen, RS Bandurski 1980 Concentration and metabolic
turnover of indoles in germinating kernels of 132_mgy§_L. Plant
Physiol 65:415-421

Labarca C, PB Nicholls, RS Bandurski 1965 A partial characterization
of indoleacetylinositols from 1ea_mgy§, Biochem Biophys Res Commun
20:641-646

Law DM, RH Hamilton 1982 A rapid isotope dilution method for analysis
of indole-3-acetic acid and indoleacetyl aspartic acid from small
amounts Of plant tissue. Biochem Biophys Res Commun 106:1035-1041

Michalczuk L, JR Chisnell 1982 Enzymatic synthesis of 5-3H-indole-
3-acetic acid and 5-3H-indole-3-acetyl-mygeinositol from
5-3H-L-tryptophan. J Labelled Compd Radiopharm 19:121-128

Mollan RC, DMX Donnelly, MA Harmey 1972 Synthesis of indole-3-acetyl
aspartic acid. Phytochemistry 11:1485-1488

Nowacki J, RS Bandurski 1980 M O-inositol esters of indole-3-acetic
acid as seed auxin precursors Of 1eg.mgy§ L. Plant Physiol
65:422-427

Nowacki J, JD Cohen, RS Bandurski 1978 Synthesis of 14C-indole-3-
acetylemygginositol. J Labelled Compd Radiopharm 15:325-329

Rittenberg 0, GL Foster 1940 A new procedure for quantitative
analysis by isotope dilution with application to the determination
of amino acids and fatty acids. J Biol Chem 133:737-744

Schlenk H, JL Gellerman 1960 Esterification of fatty acids with

diazomethane on a small scale. Anal Chem 32:1412-1414

 

20.

21.

22.

23.

24.

25.
26.

36
Sheldrake AR 1973 Do coleoptile tips produce auxin New Phytol

72:433-447

Sherman WR, NC Eilers, SL Goodwin 1970 Combined gas
chromatography-mass spectrometry Of the inositol trimethylsilyl
ethers and acetate esters. Org Mass Spectrom 3:829-840

Shvets VI 1974 The chemistry of myoinositol. Uspekhi Khim
43:1074-1101 [Russ Chem Rev 43:488-502]

Skoog F 1937 A deseeded Axeng test method for small amounts of auxin
and auxin precursors. J Gen Physiol 20:311-344

Ueda M, RS Bandurski 1969 A quantitative estimation Of alkalai-labile
indole-3-acetic acid compounds in dormant and germinating maize
kernels. Plant Physiol 44:1175-1181

Went FW, KV Thimann 1937 Phytohormones. MacMillan, New York, p 65

Williams CM, AH Porter, M Greer 1969 Mass Spectrometry of
biologically important aromatic acids. University of Florida,
Medical School and Veterans Administration Hospital Publication,

Gainesville, Florida

37

Table 1. Characteristic ions in the 70 ev mass spectra of the

m/z

103
130
157
373
547

pentaacetyl derivatives of authentic IAInos and Of IAInos

isolated from 1. mays shoot tissue.

Relative Abundance (1)

Peak 1 Peak 3 Peak 4

Authentic Isolated Authentic Isolated Authentic Isolated

4.5 3.3 5.0 3.4 9.6 3.9
100.0 100.0 100.0 100.0 100.0 100.0
45.8 41.2 74.1 69.6 76.1 66.4
- 1.5 0.5 0.8 - -

10.6 9.9 5.8 5.7 7.5 7.3

I38

 

 

 

 

 

 

 

 

om. cod x m.a No" x o.o NN.° Lou x e.~ nee_<emzmu
owm mod x m.~ cod x a.a mm cog x m.~ <<_mue~-_»uooe-~g
<<~ coumm + mock
owe so” x m.~ aoa x e.o mm mod x w.~ <<_Hua~-_xuooa-~u mmma em
<<_ ooce
em woe x o.~ No" x o.e -.o “as x 0.2 mae_<~flzmg
mac—4: N ucwetmmxm
cea «OH x ~.~ Nofi x e.e ~ao.o cos x a.m noeeaemzmu
Cam no“ x m.e mo” x m.~ as cos x ~.~ <<~n8a3-_xaooa-au
<<~ qumw + amt—u
as” ace x ~.~ moH x m." as was x ~.~ («.mua~-_»aooa-au mom m~
<<~ wwL...
so mo” x 2.3 No" x e.e ~oo.o was x m.m nae.<afixmu
mo:_<~ A ucwE_cmmxu
Hun: emote mg.»oe= _-oe:.euu Hn—oe:.eau lease Emu u: smote a a: axe m
aaam.e 8 cu eoae< aoeaa eon: etaaeaum aouoacaxm damn.»
c. ucaoe< ucsoe< »u_>_uuoo_ucm new

m:mw.u uoozm axes am :_ <<~ coumm + owe» new <<_ out» .moc_<_ we mom»_oco m>.uou_u:o=o

ammomm< uc=QEou

.u. a_noh

39

Table 111.

+ ester IAA in 1. mays shoot tissue.

The average and relative amounts Of IAInos,

free IAA and free

 

Compound

 

Amount in Tissue

nmol/kg fr. wt.
IAInos 74
Free IAA 120
Free + ester IAA 510

% Of Total

15
24
100

 

 

 

40
Figure Legends

Fig. 1. ISO chromatograms of acetylated authentic IAInos and Of acety-
lated putative IAInos isolated from 1. @215 shoot tissue. Chromatograms
A and C are the authentic and putative IAInos, respectively, from
Experiment 1. Chromatograms B and D are the authentic and putative
IAInos, respectively, from Experiment 2. The four resolvable IAInos
peaks (Pl-P4) are seen in chromatograms A and 8. Only P1 and P3 are seen
in the plant samples, chromatograms C and 0. Peaks between P1 and P3 in
chromatogram D are thought to be contaminants (see Discussion). Also
seen in chromatograms C and D is the second internal standard

bis-methyl-[14CJIAAsp (Me-IAASp).

Fig. 2. The GLC elution profiles of 3H from injections of [3H]IAInos (A)
and Of putative [3H]IAInos reisolated from 1, may; shoot tissue (B).

The 3H elution profile resulting from the injection of 460,000 dpm
(approximately 2 nmol) of acetylated [3H]IAInos diluted 1:300 with
acetylated IAInos is presented below a TSD tracing of an injection Of 1
nmol of the same sample (A). The 3H elution profile resulting from the
injection of 48,000 dpm Of acetylated putative [3H]IAInos reisolated
from the plant sample is presented below an FID tracing of a 7 nmol

injection of acetylated authentic IAInos (B).

Fig. 3. The 70 eV spectrum of pentaacetyl IAInos. Spectrum A: GLC peak
3 Of authentic pentaacetyl IAInos. Spectrum 8: GLC peak 3 of putative

pentaacetyl IAInos isolated from 1. mays shoot tissue.

 

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44
EXPERIMENTAL III

TRANSLOCATION 0F RADIOLABELED INDOLE-3-ACETIC ACID
AND INDOLE-3-ACETYL-fl0—INOSITOL FROM

ENDOSPERM T0 SHOOT 0F 1§A_MAYS L.1

2 3

John R. Chisnell and Robert S. Bandurski
Department Of Botany and Plant Pathology
Michigan State University

East Lansing, Michigan 48824-1312

Manuscript received date:

 

45
Footnotes

1Supported by grants from the Metabolic Biology Section of the National
Science Foundation, PCM 8204017, 0RD 30668, and the Life Science
Section Of the National Aeronautics and Space Administration, NAGW 97,
0RD 25796. This is Journal Article No. from the Michigan

- Agricultural Experiment Station.

“Tim's.

2Present address: Department Of MicrobiOIOQY. North Carolina State

University, Raleigh, NC 27650.

3To whom reprint requests should be directed.

4Abbreviations: IAInos, Indole-3-acetylemyQ-inositOl; MeIAA, the
methyl ester of IAA.

. 46
Introduction

It is well documented that the tip Of a growing shoot plays a
special role in regulating the growth rate Of the lower portions Of the
shoot (2,21). The studies of Went, Cholodny, Snow and others indicated
that the shoot tip supplied the growth hormone auxin (presumably
indole-3-acetic acid) to the lower portion of the shoot. However, the
exact chemistry of IAA production by the tip is unclear. It has not been

established whether the tip is synthesizing IAA g2 novo (W.L. Pengelly,

 

personal communication), or from tryptophan (9), or by hydrolysis of an

IAA conjugate (14). This work is addressed to that question by studying

 

one Of the possible precursors Of IAA for conversion by the tip.

Early work on auxins by Cholodny (4) and van Overbeek (25) 10.52202
suggested that the seed was the source Of an auxin precursor for young
seedlings. Skoog (19) developed a bioassay for a "seed auxin precursor"
in which the substance was collected into agar as a diffusate from
-decapitated coleOptile stumps. Went and Thimann (27) suggested that this
seed auxin precursor was an ester. The endosperm of kernels of 139 mgys
contains small amounts Of IAA and large amounts of esterified IAA
(1,11,16,22,23), and Sheldrake, (18) demonstrated that an alkalai-labile
IAA compound is present in the xylem sap of_1.‘mgy_, thus, potentially
being a seed auxin precursor. This laboratory has determined the
structure and concentration of all the IAA containing compounds in the 1.
Iggy; kernel,.down to a level of 10 "'g-kg-1 (see 5 for references).
IAAjflXQfTflOSTtOl (IAInos)4 comprises approximately 15% of the total IAA
of the kernel and 30% of the low molecular weight IAA esters (22). More
recently this laboratory has determined the rate of metabolic turnover Of

the indolylic compounds in the kernel (8). With this knowledge of the

47
structure, amount and turnover Of IAA compounds in the gee kernel, it was
determined from the data of Hall and Bandurski (12) that the amount Of
IAA translocated to the shoot from the kernel is insufficient to supply
the auxin needs Of the growing shoot (14). Nowacki and Bandurski (14)
using radiolabeled IAInos applied to the endosperm, demonstrated that
IAInos is translocated to the shoot and that it can be hydrolyzed in the

shoot releasing free IAA, thus qualifying it as a seed auxin precursor.

They also calculated that a sufficient amount Of ester IAA in the shoot
had arisen from IAInos in the endosperm to supply the estimated auxin )
needs of the growing shoot.

In this paper we present data on the distribution in the shoot Of

 

total radioactivity, radiolabeled IAA and radiolabeled ester IAA arising
from radiolabeled IAInos and radiolabeled IAA applied to the endosperm.
We find differences in the distribution and chemical form of the
radiolabeled IAA in the shoot depending upon whether radiolabeled IAA or
radiolabeled IAInos was applied to the endosperm. We can confirm that
IAInos applied to the endosperm provides free IAA to both the mesocotyl
and coleoptile. However, further data on amounts and sites of hydrolysis
will be required before it can be determined whether the IAInos
translocated from the endosperm is hydrolyzed in the shoot tip, thus

explaining the special role of the tip as a source of growth substance.

48

Materials and Methods
1. Radioactivity in the shoot from [14CJIAA applied to the endosperm.

Plant Material. ‘1ggimgys L. var. Stowells Evergreen Hybrid, a white
sweet-corn, was obtained from Ferry-Morse Seed Company, Mountain View,
California. Seeds were surface sterilized in 1% NaOCl for 10 min and
then soaked in running tap water (21°) for 22 to 32 h. The seeds were
rolled in paper towels, and placed in buckets, containing a small amount
of distilled water. Germination was in darkness at 25°C for 83 to 93 h
at which time the shoot mesocotyls were 4 to 8 cm and the coleoptiles 1.5
to 2.5 cm long.

Application of [14CJIAA. All manipulations were performed at 25°C with

 

the use Of a phototropically inactive green safelight. The top end Of ‘
the kernel (approximately 20% by weight) was cut off, leaving the
scutellum intact, so that an endosperm surface was exposed for isotope
application (see Figure 1). Five ul of [2-actyl-14CJIAA (51.7

mCi-nmol '1, New England Nuclear) in 50% (v/v) aqueous 2-propanol was
applied to the cut endosperm surface. Each 5 ul aliquot contained
approximately 4000 dpm equal to 35 pmol Of 14C. Radiochemical purity was
not determined, but, based upon previous experience with commercially
supplied [2-acetyl-14CJIAA, was estimated to be 80%. Thus 28 pmol Of IAA
was added to the kernel, which is equal to approximately 11% of the
endogenous IAA remaining in the kernel (8). The seedlings were pinned
through the endosperm to a styrofoam sheet with their roots dipping into
a tray of distilled water. The entire plant holder was draped with
moistened paper toweling. The seedlings (60 per time point) were
incubated for 0.5, 1, 2, and 4 h with duplicate samples used for each

point.

49

Determination of 14C in the shoot. The shoots were cut Off 0.5 cm
above the seed and dissected into 3 pieces: 1) the coleoptile, contain-
ing the primary leaves and the apical 0.5 cm of the mesocotyl, 2) the
vascular stele Of the mesocotyl (probably separated from the cortex at
the endodermis (10)), and 3) the mesocotyl cortex plus epidermis. The
pieces were placed immediately into beakers sitting on dry ice, and the

tissue was lyophilized and weighed. The samples were combusted in a

 

 

Packard model 306 Sample Oxidizer, and the 14C02 collected and counted f!
in a Packard Tri-Carb Scintillation Counter. Corrections were made for .
both combustion recovery and scintillation counting efficiency.

II.[3H]IAA, free and free + ester, translocated to the Shoot from Et

£§HJIAA applied to the endosperm. 1;
Plant Material. 1, mgys Stowells Evergreen Hybrid (Lot 80 for 1980) was
obtained from the Burpee Seed CO., Clinton, Iowa. Plant material was
grown and prepared as described in the preceeding section.

Application of [3H]IAA. [5-3HJIAA (29 Ci-nmol'l) was enzymatically
synthesized from [5-3JL-trypt0phan (29 Cionnwfl-J) (13). Radiochemical
purity was determined by the percentage of the radioactivity recoverable
from a TLC plate at the Rf Of the compound. The validity Of this
radiochemical purity determination was confirmed by reverse phase HPLC,
where the radiochemical purity equaled the percentage of the total
eluting radioactivity emerging at the elution volume for the compound.
Application of the [3H]IAA and manipulation Of the seedlings was as
described in the previous section. To one batch Of seedlings (39 in
number) 3.3 X 106 dpm (51 pmol) of [3H]IAA (at 82% radiochemical purity,
equaling 4.0 X 106 dpm 3H) in 5 ul of 50% (v/v) aqueous 2-propanol was

applied to the cut endosperm surface and the seedlings were incubated for

50
2 h. For another batch of seedlings (45 in number) 2.1 X 106 dpm (33
pmol) [3H]IAA (at 93% radiochemical purity, equaling 2.3 X 106 dpm 3H) in
2 ul of 50% (v/v) aqueous 2-propanol was applied to the cut endosperm
surface and the seedlings were incubated for 4 h. Fifty one pmol of IAA
is equal to approximately 21% of the endogenous IAA in the kernel and 33
pmol is equal to approximately 13%.

Determination of free and free + ester [3H]IAA in the shoot tissue.

The shoots were harvested and dissected as described above. The frozen if

tissue was weighed to determine the "fresh" weight and the tissue was 5

ground in sufficient acetone to result in a tissue to acetone ratio Of

30:70 (w/v). The acetone contained a known amount (about 3 umol) of IAA :
t_

 

as cold carrier. The tissue was extracted for 24 h at 23°C, then '*
filtered through tared Whatman NO. 1 filter paper, and the acetone
insoluble material dried, and used as an estimate of the dry weight Of
the tissue. Acetone was removed from the filtrate in y;ggg_and the
aqueous solution divided, one half for determination of free IAA and one
half for determination Of free + ester IAA. The free + ester samples
were made 1N with respect to NaOH and incubated for 1 h at 23°C to
hydrolyze the IAA esters. The pH of both free and free + ester samples
was adjusted to 3.5 using H3PO4, and the samples were partitioned three
times with CHCl3. The combined CHCl3 fractions were dried over NaZSO4
and evaporated to dryness ig_!gggg, The residue was dissolved in 50%
(v/v) aqueous 2-propanol and adsorbed on a DEAE Sephadex (Sigma, St.
Louis, MO) (acetate form) column (5 ml bed vol), and eluted with a linear
gradient of 50% (v/v) aqueous 2-propanol containing from 0 to 10% (v/v)
glacial acetic acid. Fractions containing IAA were identified by

Spotting 10 ul aliquots on TLC plates and staining for indoles using

51
Ehmann's Van Urk-Salkowski Reagent (7). The IAA containing fractions
were pooled, evaporated to dryness ifl ygggg, and then dissolved in
2-propanol. The sample was streaked on a 20 X 20 cm Silica Gel 60 TLC
plate (Merk) and the plate developed with CHCl3:CH3OH:H20 (85:14:1, v/v).
A guide strip from the TLC plate was stained with Ehmann's reagent, as
above, to locate the IAA. The band containing IAA was scraped Off the
TLC plate and the IAA eluted with 50% (v/v) aqueous 2-propanol. The
solution was filtered through Whatman No. 1 filter paper to remove the
silica gel, and the sample was loaded on a Sephadex LH-20 (Sigma) column
(2 ml bed volume) and eluted with 50% (v/v) aqueous 2-propanol. Frac-
tions containing IAA, identified as described for the DEAE fractions,
were pooled and evaporated to dryness in 12219, The residue was
dissolved in CH30H and methylated using ethereal diazomethane (17) and
dried under a stream of N2. The residue was dissolved in tetrahydrofuran
which had been freshly redistilled over potassium and under an N2
atmosphere. The sample was then chromatographed on 3% 0V 17 on' 60/50
Gas Chrom Q (Anspec, Ann Arbor, MI), 6 mm x 122 cm, isothermally at
188°C, using N2 as carrier gas. Substances eluting at the retention
time for MeIAA (approximately 3 to 4 min) were collected in an Open ended
glass tube plugged with silanized glass wool (Anspec). The MeIAA was
eluted from the collection tube with methanol and its UV absorbance
spectrum determined using a Cary 15 spectrophotometer. Aliquots were
taken from the spectrophotometer cuvette and counted for 3H in a Beckmann
LS 7000 scintillation counter. The shape of the UV absorbance spectrum
was used to confirm the purity Of the MeIAA collected, and its molar
extinction coefficient at 280 mm (E = 6060) was used to determine the

MeIAA concentration of the solution. The concentration of the MeIAA in

52

the solution along with the concentration of the 3H in the solution

were used to calculate the specific activity Of the reisolated IAA. The
specific activity Of the reisolated IAA multiplied by the amount Of cold
carrier IAA added equalled the amount of [3H]IAA, free or free + ester,
in the shoot at the time Of harvest.

III. [3H]IAA, free and free + ester, translocated to the shoot from
£§HJIAInOS applied to the endosperm. 1

Plant Material. Growth and preparation of the plant material was as
described previously.

Application of [3H]IAInos. [5-3HJIAInos (27 Ci/mmol) was
enzymatically synthesized from [5-3HJL-tryptophan (27 Ci/mmol, Research
Products International) by the method of Michalczuk and Chisnell (13).
The application of 4 pl aliquots Of the [3H]IAInos and incubation
conditions were as described in Section 1. Each 4 pl aliquot contained
approximately 2.2 X 106 dpm (37 pmol) of [3H]IAInos (at 83% radiochemical
purity, equaling 2.7 X 106 dpm 3H) in 50% (v/v) aqueous 2-propanol.
Thirty seven pmol equals about 1% of the endogenous IAInos in the kernel
(8). The seedlings were incubated for either 2 or 4 h. The shoots were
dissected as previously described and immediately placed in beakers
containing liquid-N2.

Determination of free and free + ester [3H]IAA in the shoot tissue.

Prior to grinding, the liquid-N2 was evaporated and the tissue
weighed to determine "fresh" weight. The tissue was ground in a glass
homogenizer with sufficient acetone to give a tissue to acetone ratio Of
3:7 (w/v). The acetone contained a known amount Of unlabeled IAA
(approximately 1.2 pmol) added as carrier. The tissue was extracted,

divided into free and free + ester samples, the free + ester sample

 

 

53

hydrolyzed, and the samples partitioned as described in Section II. The
residue remaining after evaporation of the CHCl3 layer was dissolved in
20% (v/v) aqueous ethanol containing 1% (v/v) glacial acetic acid and
placed on a Partisil 10 DDS 013 reverse phase HPLC column (0.46 X 25 cm,
Whatman) with a Co:Pell ODS precolumn (0.21 X 7 cm, Whatman). The sample_
was eluted with 20% (v/v) aqueous ethanol containing 1% (v/v) glacial

acetic acid at a flow rate Of 1 ml-min-l in a Varian 5060 liquid

chromatograph equipped with a Varian UV-5 280 nm detector. The large f-

amount of impurities prevented monitoring IAA elution by 280 nm ,

absorption, therefore, a tlc spot assay, as described in Section II, was

used to locate the IAA containing fractions. The fractions eluting at i
L.

 

the edges Of the IAA peak (less than 5% Of the peak) were discarded and
the remaining peak fractions pooled. The pooled sample was evaporated to
dryness lg ygggg and methylated using ethereal diazomethane. The
methylation reagents were evaporated under a stream of N2 and the residue
dissolved in 30% (v/v) aqeous ethanol. The sample was placed on the
reverse phase HPLC column, as previously described, and eluted with 30%
(v/v) aqueous ethanol at a flow rate of 1 ml-min-l. The IAA elutes as a
single fully resolved peak, as detected by 280 nm absorption, at
approximately 14 to 16 ml. The relative amounts of IAA in the fractions
collected at the IAA retention volumes were determined by the tlc plate
spot assay previously described. The main fractions (approxiamtely 90%
of the IAA in the peak) were pooled, the UV absorption spectrum was
determined, and aliquots were taken from the spectrophotometer cuvette
for determination of 3H by liquid scintillation counting. The amounts

Of [3H]IAA, free and free + ester, in the shoot sections were calculated

as described in Section II.

 

54

IV. Longitudinal distribution Of 3H and [3H]IAA, free and free + ester,
in the shoot translocated from [3H]IAInos applied to the endosperm.

Plant Material. Growth and preparation Of the plant material was as
described in Section 11.
Application of [3H]IAInos. TO each kernel 1.2 X 106 dpm (19 pmol) Of
[5-3HJIAInos (29 Ci/mmol; at 90% radiochemical purity, equaling 1.3 X 106
dpm 3H) in 2 pl 50% (v/v) aqueous 2-propanol was applied.
Distribution of the 3H in the shoot. Initially, a time course was run
measuring the distribution of 3H in the shoot in lots Of 10 seedlings .
incubated for 1, 2, 4, 6, and 8 h. The shoots were cut from the
seedlings approximately 0.5 cm above the kernel and then dissected into
6 pieces with 5 lateral cuts (see Figure 2A). The shoot pieces (10
pieces per vial) were placed in 20 ml scintillation vials sitting on dry
ice, and the tissue was weighed to determine the "fresh" weight. NCS
Tissue Solublilizer (Amersham) was added to the tissue, and the vials
were capped and agitated for 16 h at 45°C to digest the tissue.
Scintillation cocktail (6 g/l 2,5-diphenyloxazole, 81 mg/l 1,4
bis(2-(4-methyl-5-phenyloxazolyl))-benzene in toluene) was added to the
mixture and the sample was counted for 3H. The counting efficiency
ranged from 14 to 35% as determined by both external and internal
standards. The 4 h time point was repeated once with a slightly
different dissection as was used when the distribution of the [3H]IAA,
free and free + ester, was determined (see Figure B). In the 4 h time
point repeat experiment each shoot piece was placed in a separate vial in
order to determine the variation in translocated 3H from shoot to

shoot.

 

55

Distribution of [3H]IAA, free and free + ester. Fifty seedlings
were incubated for 4 h after which the shoots were excised and dissected
into 6 pieces as shown in figure 2B. The shoot pieces were placed in
vials sitting on dry ice and then weighed to determine the "fresh"
weight.

Determination of free and free + ester [3H]IAA in the Shoot
pieces. The procedure was as described in section 111.
V. Distribution of 3H in the kernel after incubation with applied [3H]IAA
or [3H]IAInos. 1
Ten kernels were excised from the seedlings, used for the [3H]IAA and :

[3H]IAInos translocation experiments described above. The kernels

 

‘7.

were dissected into 3 pieces Of about equal size (see Figure 1). The
uppermost section contained the cut endosperm surface to which the
labeled compound had been applied. The pieces were collected in vials
Sitting on dry ice and weighed to determine the "fresh" weight. The
tissues were then digested with NCS Tissue Solubilizer and counted for

3H as described previously.

RESULTS

The overall intent of this work is to examine 1) the special
function Of the tip of etiolated seedlings in controlling the growth Of
the subapical elongating regions, and 2) the role seed auxin precursors
play in this regulation. The purpose of the experiments presented here
was to identify the paths of translocation in the Shoot of free IAA and a
putative auxin precursor, both originating in the endosperm. TO this
end, we present data on the distribution of free and ester radiolabeled
IAA and total radiolabel in the shoot as a function of time after

applying radiolabeled IAA and IAInos to the endOSperm.

 

 

56

Experimental Design. In initial experiments a tissue oxidizer was
used in determining the total radioactivity in shoot pieces. In later
work, however, a tissue solubilizer was used because of the ease in
processing nultiple samples simultaneously.

The procedure used to determine the amount of free and free + ester
[3H]IAA required 1) that radiolabel not in IAA be removed, and 2) that
any remaining non-radiolabeled impurities not absorb significantly at 280
'Or 220 nm. The initial procedure used (Section 2), which consisted of 5 E-
purification steps (partitioning, DEAE, TLC, LH-ZO, GLC) and required 10 i

days to perform, had previously been shown to meet these criteria (14).

 

This procedure was simplified (Sections III and IV) to one involving only

1"”

3 purification steps (partitioning, and 2 reverse phase HPLC steps),
requiring 4 days to perform. The Specific activity of reisolated
[3H]IAA, as determined by the simplified procedure, did not change
significantly (less than 10%) when the IAA was further purified by GLC.
The sensitivity Of the procedure was approximately doubled by reducing,
by one-half, the amount of cold carrier IAA added. This reduction was
made possible due to the improved recovery Of IAA resulting from fewer
purification steps, and especially to the eliminatiOn Of the TLC and GLC
steps.

Raw data from experimental sections I through IV are presented in
Tables I, II, III, and IV.

Results I. Results from experimental section I are presented
graphically in figure 3 on a dpm per shoot section basis normalized to
the amount of 140 applied to the endosperm. 14C was detectable in all
parts of the shoot within one half hour, with the largest amount found in

the mesocotyl stele. When the relative dry weight ratios of stele to

 

57

cortex Dr coleoptile section (1:4) or the relative fresh weight ratios of
stele to cortex or stele to coleoptile (1:6 and 1:4, respectively) were
considered, the concentration of 14C located in the mesocotyl stele

was much higher.

At 1 h there was a 4 fold increase in the total amount of 14C in the_
shoot compared to that found at 0.5 h. The stele still contained more
14C per piece than the other shoot sections, and there was approximately
a 3 fold increase in the amount of 14C in this section over that found at
0.5 h. The coleoptile section contained about 80% as much 14C and the
cortex contained about 40% as much 14C as that found in the stele. At 2
h, there was again an approximately 4 fold increase in the total amount
of 14C in the shoot as compared to 1 h. The coleoptile section now
contained the largest amount of 14C of the 3 sections, followed by the
stele and then the cortex sections. At this time approximately 50% of
the total 14C in the shoot was located in the coleoptile section with 30%
in the stele and 20% in the cortex. In one of the 2 h incubation
experiments (I 27) the mesocotyl was not dissected. At 4 h there was a
further 1.5 fold increase in the total amount Of 14C in the shoot as
compared to 2 h. The coleoptile section still contained the largest
amount of 14C Of the 3 sections (about 55% of the total), but now the
relative amount in the cortex section had increased to about 30% of the
total shoot 14C while the relative amount in the stele section had
decreased to about 15% Of the total Shoot 14C.

Results 11. Results from experimental section II on the amount of intact
[3H]IAA, free and free + ester per shoot section, are presented in
figure 4, again, normalized to the amount of [3H]IAA applied to each

endosperm. The low radioactivities in cortex and especially in the

 

58

coleoptile sections (see Table 11) leaves Open the possibility of
inaccuracies in the amounts of [3H]IAA determined for these sections. At
both the 2 h and the 4 h time points most (85 and 80%, respectively) of
the recoverable [3H]IAA remained as the free acid, the same chemical form
as applied to the endosperm. At'2 h 85% of the [3H]IAA in the shoot was
located in the stele and 85% of this was as free [3H]IAA. This ratio of
free to ester [3H]IAA was also obtained for the cortex and coleoptile
sections, which contained 13% and 2%, respectively, Of the free + ester
[3H]IAA of the shoot. At 4 h there was a 1.5 fold decrease in the amount
of [3H]IAA, free + ester, found in the shoot contrasting sharply with a
1.5 fold increase in total radioactivity in the shoot over this time
period when [14C]IAA was applied to the endosperm. Further, there was a
redistribution Of both the location and the form (free or ester) of the
[3H]IAA from that found at 2 h. The stele section contained only about
56% of the total free + ester [3H]IAA in the shoot, while the amount in
the cortex increased to 40%. There still was only a trace amount Of
[3H]IAA found in the coleoptile section (about 4%). Only the cortex
section contained any ester [3H]IAA, present in an amount approximately
equal to the free [3H]IAA. The relative distribution in the shoot of
free + ester [3H]IAA, at both 2 h and 4 h time points, when [3H]IAA was
applied to the endosperm contrasts sharply with the relative distribution
in the shoot of total 14C when [14C]IAA was applied to the endosperm.

The most striking contrast was in the coleoptile section which contained
a large amount Of the radiolabel, from radiolabeled IAA applied to the
endosperm, but only trace amounts Of intact radiolabeled IAA. Based upon
the ratio of label recovered in the entire shoot at 2 h to that applied

to the endosperm approximately 25% of the radiolabel was located in

 

59

in intact IAA, free or ester, while at 4 h only about 10% of the
radiolabel was located in intact IAA, free or ester. Thus, at 2 h
[3H]IAA, free + ester, accounts for 70% of the 3H present in stele, 16%
in the cortex and 1% in the coleoptile. At 4 h [3H]IAA accounts for 37%
of the 3H present in the stele, 13% in the cortex and 1% in the
coleoptile.

When this data is presented on an apparent concentration basis
the relatively high amount of [3H]IAA in the stele is highlighted
(Figure 5).

Results III. The results from section III on the amount of intact
[3H]IAA, free and free + ester, per shoot piece are presented graphically
in figure 4 normalized to the amount of [3H]IAInos applied to each
endosperm. Low radioactivity in the free [3H]IAA samples, especially in
the cortex at 2 h (see Table III), make the amounts Of [3H]IAA calculated
for these sections subject to large error. When [3H]IAInos was applied
to the endosperm more than 90% of the recoverable [3H]IAA in the shoot
remained as ester IAA. In general the pattern of distribution of free +
ester and ester [3H]IAA in the Shoot was Similar tO that found for total
14C in the shoot when [14C]IAA was applied to the endOSperm (figure 4B).
The ratios of the free + ester [3H]IAA in the shoot sections were
33:22:45 (stelezcortexzcoleoptile) at 2 h and 24:32:44 at 4 h when
[3H]IAInos was applied to the endosperm compared to the ratios of 14C
found in the shoot sections of 29:21:50 at 2 h and 13:32:55 at 4 h when
[14C]IAA was applied to the endosperm. The pattern of free [3H]IAA
distribution in the shoot was markedly different from that found when

[3H]IAA was applied to the endosperm. At 2 h the ratios of free IAA in

 

 

60

the 3 shoot sections were approximately 10:1:4 (stelezcortexzcoleoptile)
and at 4 h these ratios were approximately 9:4:11 when [3H]IAInos was
applied to the endosperm. This contrasts with ratios Of 39:6:1 at 2 h
and 25:9:2 at 4 h when [3H]IAA was applied to the endosperm. The total
amounts Of both free and free + ester [3H]IAA in the shoots increased
almost 4 fold from 2 to 4 h.

When this data is presented on an apparent concentration basis
(Figure 5) both free and ester [3H]IAA are seen to have the highest
concentration in the stele, followed by that in the coleoptile section.

Results IV. The results of the experiment on the longitudinal
distribution of 3H and [3H]IAA, free and free + ester, when [3H]IAInos
was applied to the endosperm are presented graphically in figures 6, 7
and 8. The changes in the distribution and amounts of 3H per shoot
section over time are presented in Figure 6A. The total amount Of 3H
doubled from 1 to 2 h, and then increased 5 fold to peak at 4 h after
which it decreased slightly. The ratios Of the total 3H in the shoot
from 1 to 2 to 4 to 6 to 8 h was 1:2:10:9:8. The general pattern of 3H
distribution changed little after 2 h. The lower quarter of the
mesocotyl (section A) contained a large amount Of 3H. The middle two
quarters of the mesocotyl (sections B and C) contained relatively little
3H. The top quarter of the mesocotyl (sectiOn 0), including the
coleoptilar node, contained about the same amount of 3H as the lower
quarter (section A). The lower half Of the coleoptile and primary leaves
(Section E) changed the most over the time course. Starting at 2 n this
section contained approximately one half as much 3H as the upper quarter
of the mesocotyl (Section D) and progressively gained 3H until at 8 h it

was approximately equal to that in the upper quarter Of the mesocotyl.

61

The amount of 3H in the upper one half of the coleoptile and primary
leaves (Section F) increased 11 fold from 2 to 4 h then remained at
approximately the same level through 8 h.

When this data is presented on an apparent concentration basis
(figure 6B), relatively higher concentrations of 3H are seen in the
coleoptile pieces reflecting their lower "fresh" weights.

The longitudinal distribution of 3H and [3H]IAA, free and free +
ester, in the shoot at 4 h is presented in figure 7. A slightly differ-
ent dissection was used in this experiment to separate the coleoptilar
node from the mesocotyl. Considering the different dissection, the
distribution Of the average amounts of 3H in the shoot in this experiment
was very similar to that found prevously at the 6 h time point. The main
difference is the relatively lower amount of 3H in the lowest mesocotyl
section found in this experiment. In general the 3H accumulated in the
upper third of the mesocotyl to the lower half of the coleoptile
(sections C, D and E). The coleoptilar node, even though relatively
small, contains more than half as much 3H as the upper one third of the
mesocotyl and almost an equal amount of 3H to that found in the lower
half of the coleoptile and primary leaves.

This experiment was designed to determine variation from shoot to
shoot and within each shoot of the amount of 3H translocated. There were
large variations in the amount Of 3H translocated to the entire shoot
(450 to 5580 dpm) and in the distribution in the shoot. Thus, an
analysis Of variance for a randomized complete block design experiment
with 10 replications was carried out on the data to determine the
significance Of the differences in amount of 3H in the shoot sections.

The LSDO.1 value indicates that section C contained significantly more

 

 

62

3H than the other sections, and that sections 0 and E contain
significantly more 3H than sections A, B, and F.

In general the distribution of free + ester [3H]IAA reflects that
found for 3H (Figure 7). In the whole shOot [3H]IAA, free + ester,
accounts for approximately 35% of the 3H. Within the shoot pieces the
notable exceptions are the coleoptilar node (section 0) where [3H]IAA,
free + ester, accounts for only 25% of the 3H present, and in the upper
half of the coleoptile and primary leaves (section F) where [3H]IAA, free
+ ester, accounts for almost 50% Of the 3H present. As was found in the
stele, cortex, coleoptile dissection, free [3H]IAA in the whole shoot
accounts for approximately 9% of the free + ester [3H]IAA. Within the
shoot sections variations from this average were found in the lower third
of the mesocotyl (section A, 21%) and the upper third of the mesocotyl
and the upper half Of the coleoptile and primary leaves (sections C and
F, respectively, both 5%).

When the results for the [3H]IAA, free and free + ester, and the 3H
are presented on an apparent concentration basis (Figure 8) the only
relative differences from the dpm per section results appear in the
coleoptilar node section and the coleoptile and primary leaves sections
(sections 0, E and F). The apparent free + ester [3H]IAA concentrations
in sections C, D and F were approximately the same and approximately 3
fold higher than that found in sections A and B and 20% less than that
found in section E. The relative apparent concentrations of 3H in the
shoot pieces were similar to those found previously at the 6 h time point
where the 3H was most concentrated in the upper half of the shoot. The
coleoptilar node (section 0) is unique in having the highest apparent

concentration of 3H.

 

1W

 

63

Results V. The results of the distribution of 3H in the corn kernel
after incubation for 2 to 8 h with [3H]IAA or [3H]IAInos are presented in
Table V. There was essentially no difference in the distribution of 3H
in the seed whether the seed was incubated for 4 or 8 h, or whether
[3H]IAA or [3H]IAInos was applied to the seed. The average ratio of the
3H in the seed pieces at 4 and 8 h was 85:13:2, top to middle to bottom
pieces. In the one experiment, where the seeds were incubated for only 2
h with [3H]IAA, relatively more radioactivity was located in the top
piece. The ratios Of 3H in the top to middle to bottom pieces were
97:7:1.

In three of the 3H distribution in the seed experiments the %
recoveries of 3H applied to the seeds were significantly greater than
100% (III 45, III 59 and III 101). However, the distribution of 3H in
these experiments was the same as that found in the other experiments
where the % recoveries were either less than 100% or, within experimental
error, essentially equal to 100%. The high recoveries may have been due

to chemiluminescence.

Discussion

It is well established that the tip of a plant serves as‘a source of
IAA for the subapical portions. For example, the tip contains more IAA
than lower coleoptile sections, as determined by bioassay (20,24) and
cessation of growth of the coleoptile and mesocotyl due to decapitation
can be reversed by exogenous IAA (6,23). It is not known, however,
where the IAA in the tip comes from. In an etiolated seedling, all Of
the compounds Of the shoot must have originated in the seed since the

seedling is not photosynthetic. But, the chemical form of the

r-i

 

 

64

substance(s) moving from the seed to the shoot have not been extensively
studied. Thus we do not know whether tryptOphan is translocated from the
seed to the shoot tip and there converted to IAA; whether simple
non-aromatic precursors are translocated to the shoot tip for gglppxp IAA
synthesis, or whether IAA conjugates move from the seed to the Shoot tip
and are hydrolyzed to yield free IAA.

Our initial hypothesis in these studies was based upon the classic
concept of auxin relationship in young seedlings in which the seed auxin
precursor moves to the tip of the shoot where is is converted to an
active auxin, and then transported to the growing region where hormonal
control is expressed (27). Went and Thimann (27) suggested that this
seed auxin precursor is an ester. That the seed auxin precursor in 1,
‘pgyg is an 1AA ester is supported by Sheldrake's observation Of an
alkalai-labile IAA compound in the xylem sap (18).

Previous work in this laboratory has identified the structure and
concentrations of all the IAA containing compounds of the 1. mgy§_kernel
down to a level of 10 pg-kg-1 (see 5 for references). IAInos comprises
about 15% of the total IAA and 30% of the low molecular weight IAA esters
of the endosperm (22) and has been calculated to be translocated from the
endosperm to the shoot in sufficient amounts to serve as a seed auxin
precursor (14). It has also been demonstrated that insufficient amounts
Of free IAA were translocated from the endosperm in 1, mgyg to supply the
auxin needs of the growing shoot (12,14).

The data of this paper, using the assumptions of Nowacki and
Bandurski (14) (that there is complete equilibration of applied
[3H]IAInos with the endogenous IAInos and that the half-life of the

IAInos pool in the 1g; kernel is 12 h, as determined by Epstein et al.

 

65

(8)), indicates that IAInos in the endOSperm was supplying approximately
1 to 2 pmol of IAA, free + ester, per h to each shoot. That IAInos
supplies 1 to 2 pmol IAA, free + ester, per h is in fairly close
agreement with Nowacki and Bandurski's calculation of 6.3 pmol-hr1 and
would supply 10 to 40% of the estimated 5 to 9 pmol-h“1 needed per shoot
(14). Data from other experiments (to be published elsewhere) indicate
that IAInos accounts for approximately 17% of the ester IAA of the shoot.
Thus, it is possible that other IAA esters are also serving as seed auxin
precursors and account for our, less than sufficient transport rate of
IAA, free + ester, from IAInos in the kernel. Most of the [3H]IAA, free
+ ester, in the shoot was located in the coleoptile plus primary leaves
and the upper one third of the mesocotyl (regions of higher growth rate
(15)) when [3H]IAInos was applied to the endosperm. This contrasts
Sharply with the only trace amounts of [3H]IAA, free + ester, found in
the coleoptile when [3H]IAA was applied to the endosperm, thus supporting
the hypothesis that IAA in the shoot tip arises from ester IAA in the
endosperm and not from free IAA in the endosperm. However, the two
incubation times chosen for these experiments in which [3H]IAA was
reisolated from the shoots do not allow us to determine if the [3H]IAInos
translocated to the coleoptile was hydrolyzed to free [3H]IAA and then
translocated basipetally to the growing region. Because of the high
concentrations of ester [3H]IAA in the vascular stele it is likely that
it was being translocated in this tissue. Large amounts of ester [3H]IAA
were translocated to the coleoptile plus primary leaves section of the
shoot but the percent free [3H]IAA in this section was only

intermediate between that found for the mesocotyl stele in the mesocotyl

cortex. This intermediate level may reflect the mix of the mostly

 

66

non-vascular tissue of the coleoptile and the large amount of vascular
tissue in the primary leaves of this Shoot section. In addition, our
data on the uniformity of the longitudinal distribution of free [3H]IAA
in the shoot 4 h after [3H]IAInos was applied to the endosperm is not in
agreement with the hypothesis that the ester IAA should be hydrolyzed to
free IAA in the shoot tip. However, if experiments with longer
incubation times are performed, a wave of free [3H]IAA moving down from
the shoot tip might be observed. Also, if the coleoptile were separated
from the primary leaves it might allow the observation of the hydrolysis
Of translocated ester IAA in the coleOptile tip that otherwise might be
obscured by potentially high concentrations of translocated radiolabeled
IAA in the vascular tissue of the primary leaves.

Radiolabeled IAA, free and ester, assumed strikingly different
proportions and distributions in the 4 day old etiolated 1. mpys shoot
depending upon whether they were derived from radiolabeled IAA or
radiolabeled IAInos applied to the endosperm. However, total
radioactivity in the Shoot derived from either radiolabeled IAA or
radiolabeled IAInos applied to the endosperm was distributed in
essentially the same manner. Free plus ester radiolabeled IAA in the
shoot derived from radiolabeled IAInos applied to the endosperm was
distributed in approximately the same pattern as the total radioactivity
in the shoot. There was little conversion from free to ester (15 to 20%)
and even less from ester to free (5 to 10%) Of the applied radiolabeled
compounds. The radial distribution of free [3H]IAA, at both 2 and 4 h,
between the mesocotyl vascular stele and the mesocotyl cortex-epidermis,
whether it came from [3H]IAA or [3H]IAInos applied to the endosperm,

approximately reflects that found for endogenous free IAA (15). That

 

 

67

most of the free IAA is localized in the vascular stele, whereas the
surrounding cortex-epidermis contains very low levels, has also been
demonstrated in 1, mays roots (3,10). The distributions of ester [3H]IAA
did not reflect that found for endogenous ester IAA. In all of the
translocation experiments, except one, the relative concentrations of
ester [3H]IAA were much higher in the stele than the cortex as compared
to the approximately 1:1 ratio of the concentrations of endogenous ester
IAA (15). However, it is possible that insufficient time had elapsed to
allow the translocated ester [3H]IAA to equilibrate with the endogenous
ester IAA in the shoot. In the [3H]IAInos experiments the ratio of the
amount of ester [3H]IAA in the stele to that in the cortex-epidermis
increased from approximately 1.5:1 to 1:1.5, and with longer incubation
times might continue to increase to the 1:15 ratio found for the
endogenous ester IAA.

The longitudinal distribution of free [3H]IAA and ester [3H]IAA in
the shoot, 4 h after the application of [3H]IAInos t0 the endosperm,
reflected an opposite trend to that found for endogenous free IAA and
ester IAA (15). Free [3H]IAA was fairly evenly distributed up the shoot
contrasting with the 2 to 3 fold higher concentration of endogenous free
IAA in the apical one fourth of the mesocotyl. Ester [3H]IAA was almost
3 fold more concentrated in the apical one third of the mesocotyl
contrasting with a uniform concentration along the mesocotyl of
endogenous ester IAA.

Further experiments are also needed to investigate the apparent
cessation of the translocation of applied radiolabeled compounds fran the
endosperm to the shoot after approximately 4 h. This was observed in the

translocation Of total radioactivity when [3H]IAInos was applied to the

 

 

68

endosperm. Since only approximately 0.2% of the applied radioactivity
had translocated to the shoot the remaining radiolabeled compounds may
have been metabolized to compounds which are not translocated or in some
other way immobilized in the kernel.

It is obvious from the above discussion that a very rapid method of
measuring, and determining the Specific activity of IAA and its
conjugates is needed to continue and complete this investigation. Only
in that way can sufficient time points be established to accurately
reflect the identity, source, routes of translocation and turnover rates
of the possible IAA precursors. Despite the paucity of data, we wish to
propose two working hypotheses that are consistent with our present
knowledge. In the first hypothesis, at least some of the IAA of the
Shoot originates in the seed as IAInos. This ester moves in the vascular
system - probably the xylem - to the apex of the mesocotyl and the tip of
the coleoptile. At these points, a portion of the easter is hydrolyzed
to free IAA, and the free IAA begins its downward movement still in the
vascular bundle but now in or near the phloem (26). Most of the free IAA
now leaks from the vascular bundles in the coleoptile and the stele in
the mesocotyl into the surrounding cortex. In the cortex the IAA is
either quickly used up in the growth promoting reaction or it is
reconjugated. The special function of the coleoptile tip and the
mesocotyl apex may lie in serving as crossover points from xylem to
phloem and as points of conjugate hydrolysis.

In our second working hypothesis, again, some of the IAA of the
shOot originates in the seed as IAInos and moves to and through the shoot
in the vascular system - probably the xylem. However, as the IAInos

moves up the shoot it leaks out, from the mesocotyl stele into the

 

69

mesocotyl cortex and from the vascular bundles into the coleoptile
cortex, in proportion to other metabolites as needed for growth in that
region. The IAInos in the cortical tissue could then serve as an IAA
precursor being converted to IAA as needed.

The data in this paper indiCates that free IAA in the seed is the
source of some of the IAA of the mesocotyl but that IAA from the
endosperm supplies essentially none of the IAA in the coleoptile or
primary leaves. Further, the coleOptilar node appears to be a gate ’
closed to the acropetal movement of free IAA. These conclusions are I
incorporated into the above two hypotheses.

The above are simply working hypotheses and they are complex.
However, the hypotheses are subject to experimental tests, and we believe
that ultimately a better understanding of the regulation of hormone

concentrations will result.

3.

4.

5.

6.

9.

10.

70

Literature Cited

Berger J, GS Avery 1944 Isolation of n auxin precursor and an auxin
(indoleacetic acid) from maize. Am J Bot 31:199-208

Boysen-Jensen P 1936 Growth hormones in plants. McGraw-Hill, New
York

Bridges IG, JR Hillman, MB Wilkins 1973 Identification and
localization of auxin primary roots of 1ggflmgy§ by mass
spectrometry. Planta 115:189-192

Cholodny N 1935 Uber das keimungshormon von gramineen. Planta
23:289-312

Cohen JD, RS Bandurski 1982 Chemistry and physiology of the bound
auxins. Ann Rev Plant Physiol 33:403-430

Dolk HE 1936 GeotrOpism and the growth substance. Rec Trav Bot Neerl
33:509-585

Ehmann A 1977 The Van Urk-Salkowski reagent - a sensitive and
Specific chromogenic reagent for silica gel thin-layer
chromatographic detection and identification of indole derivatives.
J Chromatogr 132:267-276

Epstein E, JD Cohen, RS Bandurski 1980 Concentration and metabolic
turnover of indoles in germinating kernels of 1eg,mgys_L. Plant
Physiol 65:415-421

Gordon SA 1956 The biogenesis of natural auxins. .15 RL Wain, F
Wightman, eds, The chemistry and mode of action of plant growth
substances, Academic Press, New York, pp 65-75

Greenwood MS, JR Hillman, 5 Shaw, MB Wilkins 1973 Localization and

identification of auxin in roots of 1gp may . Planta 109:369-374

 

 

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

71

Haagen-Smit AJ, WD Leech, WR Bergen 1942 The estimation, isolation,
and identification of auxins in plant materials. Am J Bot
29:500-506

Hall PL, RS Bandurski 1978 Movement of indole-3-acetic acid and
tryptophan-derived indole-3-acetic acid from the endosperm t0 the
shoot of 1;; mgys L. Plant Physiol 61:425-429

Michalczuk L, JR Chisnell 1982 Enzymatic synthesis of 5-3H-indole-
3-acetic acid and 5-3H-indole-3-acetyl1mygeinositol from F‘
5-3H-L-trypt0phan. J Labelled Compd Radiopharm 19:121-128

Nowacki J, RS Bandurski 1980 M o-inositol esters of indole-3-acetic

 

acid as seed auxin precursors Of 121 may; L. Plant Physiol 3
65:422-427

Pengelly WL, PJ Hall, A Schulze, RS Bandurski 1982 Distribution of
free and ester indole-3-acetic acid in the cortex and stele of the
1gghmgy§ mesocotyl. Plant Physiol 69:1304-1307

Piskornik Z 1975 Distribution of bound auxin in kernels of seet corn
(1egumgys L). Acta Biol Cracoviensa Bot Ser 18:1-12

Schlenk H, JL Gellerman 1960 Esterification of fatty acids with
diazomethane on a small scale. Anal Chem 32:1412-1414

Sheldrake AR 1973 Do coleoptile tips produce auxinn New Phytol
72:433-447

Skoog F 1937 A deseeded Agegg test method for small amounts Of auxin
and auxin precursors. J Gen Physiol 20:311-334

Thimann KV 1934 Studies on the growth hormone of plants VI. The
distribution of the growth substance in plant tissues. J Gen

Physiol 18:24-34

 

21.

22.

23.

24.

25.

26.

27.

72

Thimann KV 1977 Hormone action in the whole life of plants. The
University of Massachusetts Press, Amherst

Ueda M, RS Bandurski 1969 A quantitative estimation of alkalai-labile
indole-3-acetic acid compounds in dormant and germinating maize
kernels. Plant Physiol 44:1175-1181

van Overbeek J 1936 Growth hormone and mesocotyl growth. Rec Trav
Bot Neerl 33:333-340

van Overbeek J 1938 Auxin distribution in seedlings and its bearing
on the problem of bud inhibition. Bot Gaz 100:133-166

van Overbeek J 1941 A quantitative study of auxin and its precursor

 

in coleoptiles. Am J Bot 28:1-10
Wangermann E, LA Withers 1978 Auxin transport characteristics and
cellular ultrastructure of different types of parenchyma. New
Phytol 81:1-17
Went FW, KV Thimann 1937 Phytohormones. MacMillan, New York
(republished 1978 Allanheld, Osmun a Co, Montclair) p 65

.mPQEom con mu_e= mo commmcaxm m. cacao

 

73

 

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75

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78

Figure Legends

Fig. 1. Diagram of a 4-d-old dark grown ggg mays seedling. The location
of the endosperm surface exposed for isotope application, and the

dissection of the kernel for experimental section V are shown.

Fig. 2. Location of the tissue sections used in the longitudinal
distribution experiments. The dissection used in determining the
distribution of total radioactivity as a function of time (Fig. 6) is
indicated in figure 2A. The dissection used in determining the
distribution of [3H]IAA, free and ester, and total radioactivity at 4 h

(Fig. 7) is indicated in figure 28.

Fig. 3. The distribution of radioactivity in the shoot as a function of
time after application of [14CJIAA to the endosperm. The values at

each time point, except 2 h which was not repeated, are the average of
two determinations. The data are normalized to the amount of 14c
applied per endosperm. St = mesocotyl stele, Cr = mesocotyl cortex +
epidermis, and Cl = apical 0.5 cm of the mesocotyl and the coleoptile

containing the primary leaves.

79
Fig. 4. The distribution of [3H]IAA, free and ester, in the shoot 2 h
and 4 h after the application of [3H]IAA and [3H]IAInos to the endosperm.
The values, when [3H]IAInos was applied, are the average of two
determinations. The data are normalized to the amount of [3H]IAA or
[3H]IAInos applied per endosperm. The amount of ester [3H]IAA was
calculated by subtracting the amount of free [3H]IAA determined from
the amount of free + ester [3H]IAA determined. St = mesocotyl stele,

Cr ' mesocotyl cortex + epidermis, and Cl = apical 0.5 cm of the

mesocotyl and the coleoptile containing the primary leaves.

Fig. 5. The concentration of [3H]IAA, free and ester, in the shoot 2 h
and 4 h after the application of [3H]IAA and [3H]IAInos to the endosperm.
The values, when [3H]IAInos was applied, are the average of two
determinations. The data are normalized to the amount of [3H]IAA or
[3H]IAInos applied per endosperm. The amount of ester [3H]IAA was
calculated by subtracting the amount of free [3H]IAA determined from

the amount of free + ester [3H]IAA determined. St = mesocotyl stele,

Cr = mesocotyl cortex + epidermis, and Cl = apical 0.5 cm of the

mesocotyl and the coleoptile containing the primary leaves.

Fig. 6. The longitudinal distribution of radioactivity (A) and
concentrations of radioactivity (B) in the shoot as a function of time
after application of [3H]IAInos to the endosperm. Shoot dissection

(tissue pieces A-F) was as indicated in figure 2A.

80
Fig. 7. The longitudinal distribution of [3H]IAA, free and ester, and
total radioactivity in the shoot 4 h after application of [3H]IAInos to
the endosperm. Shoot dissection (tissue pieces A-F) was as indicated in

figure 28. L.S.D. value at the 0.1 level for total radioactivity = 192.

Fig. 8. The longitudinal distribution of the concentration of [3H]IAA,
free and ester, and total radioactivity in the shoot 4 h after
application of [3H]IAInos to the endosperm. Shoot dissection (tissue

pieces A-F) was as indicated in figure 28.

81

)-

/y/COLEOTPTILE CONTAINING
THE PRIMARY LEAVES

I 1’1—-C0LE0PTILAR NODE

 

 

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PORTION OF KERNEL
REMOVED FOR LABELED
COMPOUND APPLICATION
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PER EXPERIMENTAL
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CONCLUSIONS

At the outset of this study the working hypothesis in use was
based upon the classic concept of auxin relationships in young seedlings
in which the seed auxin precursor moves to the tip of the Shoot where it
is converted to an active auxin, and then transported to the growing
region where hormonal control is expressed. It had been proposed that
IAA-m o-inositol was a seed auxin precursor in the Shoots of young dark-
grown Z, mgy§_seedlings. This was based upon the observations that 1)
high concentrations of IAA-m o-inositol are found in the kernel (com-
prising about 15% of the total IAA), 2) that radiolabeled IAA-m o-
inositol can be translocated from the endosperm to the shoot and there
be hydrolyzed releasing IAA, and 3) the calculation that IAA-myg:
inositol in the kernel supplied a sufficient amount of ester IAA to the
shoot to serve as a seed auxin precursor.

In further investigating the proposition that IAA-m o-inositol
is a seed auxin precursor in the shoots of young dark-grown Z;_mgy§_this
study has established that IAAam o-inositol is an endogenous component
of the Shoot tissue. It is present in the five-day-old shoot at a
concentration of approximately 74 nmole per kg fresh weight and accounts
for about 15% of the free + ester IAA or 19% of the ester conjugated IAA
found there. This ratio of IAA-m o-inositol to ester conjugated IAA
is Similar to that foundirIthe kernel after four days of germination
(25%) (Epstein et al., 1980). This study also provides data on the

89

9O

translocation of radiolabeled IAA-mygrinositol and radiolabeled IAA
from the endosperm to the shoot and the subsequent distribution of free
and ester radiolabeled IAA in the shoot. The estimate in this study
that IAA-m o-inositol in the endosperm supplies 1 to 2 pmole free +
ester IAA to each shoot per hour is in fairly close agreement with
Nowacki and Bandurski's (1980) calculation of 6.3 pmole per hour. The
calculated transport rate of this study would be sufficient to supply
10 to 40% of the estimated 5 to 9 pmole needed per hour per Shoot. As
indicated above, IAA-mygyinositol accounts for only 19% of the endoge-
nous ester conjugated IAA in the shoot. Thus. it is possible that other
IAA esters are also serving as seed auxin precursors and account for
the less than sufficient transport rate of free and ester IAA from IAA-
m o-inositol in the kernel.

Radiolabeled IAA and radiolabeled ester IAA assumed strikingly
different proportions and distributions in the shoot depending upon
whether they were derived from radiolabeled IAA or radiolabeled IAA-
mygyinositol applied to the endosperm. Both compounds appeared to be
transported, at least initially, in the vascular stele of the mesocotyl.
Radiolabeled free and ester IAA in the shoot, derived from radiolabeled
IAA applied to the endosperm was almost entirely restricted to the
mesocotyl. The coleoptilar node appeared to be a gate closed to the
acropetal movement of IAA. This was in Sharp contrast to when radio-
labeled IAA-m o-inositol was applied to the endosperm, more than one-
third of the free and ester IAA in the shoot was found at or above the
coleoptilar node in the coleoptile and primary leaves. There was little
conversion from free to ester (15 to 20%) and even less from ester to

free (5 to 10%) of the applied radiolabeled compounds.

91

In order to accomplish the above work a synthesis for high
specific activity tritiated IAA and IAA-m o-inositol was developed.
This was an enzymatic synthesis on a nanomolar scale with little or no
dilution of the initial specific activity of the L-[5-3H]tryptophan
substrate.

Two working hypotheses are presented which incorporate the data
of this study and hopefully point the direction for further research on
the source of IAA in etiolated seedlings. There are a number of I

questions which need to be investigated in the future. First, a much

 

more rapid and possibly more sensitive method of measuring and deter-
mining the specific activity of IAA and its conjugates needs to be
developed. This would enable the analysis of an increased number of
time points and dissections of the Shoot, which are required to accu—
rately determine the identities, sources, routes of translocation,
locations and turnover rates of the possible IAA precursors. Second,
the identities and concentrations of the remaining 80% of the endog-
enous IAA esters in the shoot need to be determined. Also, it would be
interesting to investigate the apparent cessation of the translocation
of applied radiolabeled compounds approximately four hours after appli-
cation. Finally, the question of which of the IAAgmyg-inositol isomers
are involved in the regulation of IAA levels and the sites of their
hydrolysis needs to be determined. This work is currently being
pursued by Hall and Bandurski (1981, 1983) who have demonstrated IAA-
m o-inositol hydrolysing activity in a partially purified enzyme prepar-

ation from dark-grown ;;_mays shoots.

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