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Michigan St". re
University

This is to certify that the

thesis entitled

STUDIES OF INDOLYLIC COMPOUNDS
OF ORYZA SATIVA

 

presented by

Prudence Ann Hall

has been accepted towards fulfillment
of the requirements for

 

M.S. I dggreein Botany and
Plant Pathology

hhmnpnmuun

Datew

0.7639

 

 

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STUDIES OF INDOLYLIC COMPOUNDS 0F ORYZA SATIVA

 

By

Prudence Ann Hall

A THESIS

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

MASTER OF SCIENCE

Department of Botany and Plant Pathology

1979

ABSTRACT

STUDIES OF INDOLYLIC COMPOUNDS 0F ORYZA SATIVA

 

By

Prudence Ann Hall

Some indolylic compounds of Oryza sativa have been assayed.

 

0f the 2200 pg/kg esterified indole-3-acetic acid, over 90 percent
appears to be present in a high molecular weight fraction. One
low molecular weight ester, indole-3-acetyl-my9:inositol, was
purified by the following sequence of chromatographic steps:
column chromatography over Dowex 50, high pressure liquid chroma-
tography over Beckman PA 28, preparative thin-layer chromatography
on Silica Gel 60, and column chromatography over Sephadex LH-20.
The ester was characterized by its chromatographic properties on
thin-layer plates; by the retention time of its trimethylsilyl
derivative in a gas chromatograph; by the products of ammonolysis,
indole-3-acetic acid, indole-3-acetamide, and mygfinositol; and by
the fragmentation pattern 0f its trimethylsilyl derivative using
combined gas chromatography-mass spectrometry. The partial charac-

terization of free tryptOphan is also reported.

ACKNOWLEDGEMENTS

I would like to acknowledge the interest and encouragement
of Dr. Robert S. Bandurski, my major professor. The assistance
and interest of Dr. Norman Good and Dr. Derek Lamport, members of
my guidance committee, is also appreciated. Dr. Richard K. Chapman
of the Michigan State University Mass Spectrometry Facility was a
patient teacher. I would also like to acknowledge the willing

assistance and helpful comments of Dr. Jerry Cohen.

ii

TABLE OF CONTENTS

LIST OF TABLES .
LIST OF FIGURES
LIST OF ABBREVIATIONS

INTRODUCTION
MATERIALS AND METHODS

Plant Materials

Chromatographic Materials

Chemicals

RadioisotOpe Materials . .

Preparation of Crude Extracts .
Chromatrographic Procedures . .
Determination of the Amounts of Free and Total IAA.
Spectrosc0py

Ammonolysis . . -
Derivatization of Compounds for CC and GC- MS .
GS -MS. . . . . . .

RESULTS

Quantitative Determination of Free and Total IAA in
O. sativa . . . . .

Isolation of Esters of IAA From 0. sativa .

Ammonolysis . .

Characterization of the 0. sativa Ester by GC .

Characterization of the O'. sativa Ester by GC -MS .

Isolation and Partial Characterization of TryptOphan
from Q_. sativa . . . . . . .

DISCUSSION

Quantitative Determinations of IAA

Isolation and Purification of Esters

Esters of IAA in Husks of 0. sativa . . .
Mass Spectrometry and Interpretation of Spectra .
Free Tryptophan in Q_. sativa . .

Page

vi

vii

—-l

O 00mmVO‘U'IU‘IUW-b-D- ->

Page
CONCLUSIONS . . . . . . . . . . . . . . . . . 44
REFERENCES . . . . . . . . . . . . . . . . . 47

iv

Table

LIST OF TABLES

Results of the Quantitative Determination of Free
and Total IAA in Q, sativa. .

Gas Chromatography of the TMS Derivatives of the
Products of Ammonolysis . . . . .

Gas Chromatography of the TMS Derivatives of IAA-
Inositol and of the Rice Ester . .

Relative Abundancies of Ions in the Mass Spectra of
the TMS Derivatives of Authentic IAA-Inositol and
IAA-Inositol Purified from Q, sativa . . .

Free and Total Inositol in Seeds

Page

ll

l8

20

4O
46

Figure

LIST OF FIGURES

Procedure for the purification of IAA Esters .

Elution pattern from Dowex 50 of a crude extract of
Q, sativa

IAA ester preparation from 0. saLiva after
chromatography over Dowex 50 and Beckman PA 28

Thin-layer chromatogram of the products of
ammonolysis . . . . . . .

Mass spectra obtained with the DP 102 GC- MS of the
axial ester of IAA- inositol

Mass spectra obtained with the UP 102 6C -MS of the
equatorial ester of IAA- inositol . .

Thin- layer chromatogram of indoleacetylaspartate,

tryptophan, tryptophan from 0. sativa, and trypta-

mine, developed in G solvent

Visible light sepctra of authentic L-tryptophan and
tryptOphan from Q, sativa after Ehmann assay .

Ultraviolet Spectra of authentic L- tryptophan and
tryptOphan from 0. saLiva .

vi

Page
12

13

15

T7

2T

23

26

27

28

amu

BSTFA

DP l02 GC-MS
GC

GC-MS

HP 5985 GC-MS
HPLC

IAA
IAA-inositol
Indoleaceamide
Mt

m/z

TL

TLC

TMS

TMSOH

UV

LIST OF ABBREVIATIONS

atomic mass units
Bis-(trimethylsilyl)trifluoracetamide

DuPont 102 Gas Chromatograph-Mass Spectrometer
gas chromatography

gas chromatography-mass spectrometry
Hewlett-Packard Gas Chromatograph-Mass Spectrometer
high pressure liquid chromatography
indole-3-acetic acid

indole-3-acetyl-m o-inositol
indole-3-acetamide

molecular ion

mass to charge ratio

thin-layer

thin-layer chromatography

trimethylsilyl

trimethylsilanol

ultraviolet

vii

INTRODUCTION

Conjugates of indole-3-acetic acid (IAA) are found in all
monocotyledonous and dicotyledonous plants that have been examined
(6). In cereal grains, esterified IAA is the predominant conjugate
and is present in greater concentration than free IAA. For example,
in seeds of Zga_may§_var. Stowell's Evergreen there is 500-100 ug/kg
of free IAA and 70-80 mg/kg of esterified IAA. Thus less than one
percent of the total IAA is present as the free acid.

IAA esterified to mygfinositol is the major IAA ester found
in sweet corn kernels. It is also found in other corn varieties,
field corn and popcorn, and in some other members of the subfamily
AndrOpogineae such as Teosinte, Tripsacum and Coix (Job's Tears)

(A. Ehmann, personal communication). IAA-inositol, IAA-inositol-
arabinosides, and IAA-inositol galactosides account for nearly all
the low molecular weight esters found in sweet corn kernels (l5);
low molecular weight esters constitute 50 percent of the total
acetone extractable esters of IAA.

The demonstration of the presence of esters of IAA and their
subsequent chemical characterization (l6,l8,l9,29,37,53,54) has
allowed studies of their role in plant growth. Recent work from
this laboratory demonstrates that IAA esters are involved in the

regulation of hormone concentration (4,7). The particular ester,

IAA-m o-inositol (IAA-inositol), is a seed auxin-precursor (2l,25,38)
and plays a role in the transport of IAA. Nowacki and Bandurski (38)
have shown that 14C-IAA-inositol applied to the cut endosperm of a
corn kernel is transported to the shoot. IsotOpe dilution studies
further demonstrate that the rate of transport of the ester is suffi-
cient to provide the shoot with the IAA that it needs for growth,
whereas Hall and Bandurski (25) have shown that free IAA is not
transported from seed to shoot at a rate that will provide the amount
of IAA calculated to be necessary for growth. Esterification of IAA
to mygfinositol protects the indole moiety from oxidation by
peroxidase (l2).

Since much of the endogenous IAA is esterified in Zga_and in
other cereal grains, and because in Zgg_esters of IAA play important
physiological roles, it was of interest to determine which esters
of IAA occurred in another cereal grain. A member of the Gramineae

only distantly related to Zga_was chosen. Rice (Oryza sativa) is

 

classified in a different grass subfamily, the Oryzoideae (22).
Morphologically corn and rice are quite distinct; furthermore, they
came under cultivation as grain crops in different hemispheres at
different times, and methods of cultivation are very different (1,34).
One low molecular weight ester of IAA from rice grains was
isolated and identified as IAA-inositol. The criteria for its
identification are Rf on thin-layer chromatography (TLC), the reten-
tion time of the trimethylsilyl (TMS) derivative by gas chromatography
(GC), the products of ammonolysis, and the fragmentation pattern of

the TMS derivative using combined gas chromatography-mass spectrometry

(GC-MS) as compared to derivatized authentic synthetic IAA-inositol.
There are indications that the bulk of the esterified IAA in rice

'kernels is a high molecular weight ester as is also the case in Zea

(4T) and Avena (40).

MATERIALS AND METHODS

Plant Materials

 

Seeds of Q, satjyg_var. Calrose from the Sutter County,
CA 1976 harvest were kindly provided by Mr. Herman Cohen of the USDA
at Sacremento, CA.‘ A commercial variety of rice, Comet Brown Rice,
Houston, TX, was purchased locally for use in early experiments.

Seeds of Avena sativa var. Ausable from the Ingham County, MI l978

 

harvest were provided by the Department of Crop and Soil Science,
Michigan State University, East Lansing, MI. Seeds of Zga_mays var.
Stowell's Evergreen were purchased from Vaughan Jacklin Corp. Ovid,

MI.

Chromatographic Materials

 

Thin-layer (TL) chromatographs were run on Silica Gel 60 TL
plates without fluorescent indicator (E. Merck, Darmstadt, Germany).
Dowex 50X2-400 (Sigma) was prepared in the Na+ form and suspended in
50 percent aqueous 2-propanol. The Beckman PA 28 resin (Beckman)
was used in a high pressure liquid chromatography (HPLC) system
developed and described by Cohen and Bandurski (ll). Sephadex LH-20
and DEAE-Sephadex were purchased from Sigma. A Varian Series 2700
GC and a Hewlett Packard 402 GC were used employing flame ionization

detectors. OV-l on Gas-Chrom Z (TOO/120) was purchased from Applied

Science Laboratories, State College, PA. The 0V-lOl capillary column

was prepared by Dr. R. K. Chapman.

Chemicals

IAA, m o-inositol and L-tryptOphan were purchased from Sigma.
Bis-(trimethylsilyl)trifluoroacetamide (BSTFA) with 1 percent
trimethylchlorosilane was purchased from Regis Chemical Co.;
dimethylformamide was purchased from Fisher ACS. Indole-3-acetamide
(indoleacetamide) was obtained from a collection prepared by Dr. A.
Ehmann. IAA-inositol, synthesized by the method of Nowacki et al.
(39), was a gift from J. 0. Cohen.

Radioisotope Materials
[214CJ-IAA was purchased from New England Nuclear Co., and
radioactivity determined with a Packard Tri-Carb Liquid Scintillation

Spectrometer using ACS solution (Amersham) as the scintillant.

Preparation of Crude Extracts
Seeds of Q, sativa_and A, saLiva_were dehusked by brief
grinding in a Waring Blendor and the husks separated from the seed
with an air stream. In the case of Q, sativa, dehusked seeds were
hand selected to insure that the preparation contained husk-free
grains. The A, sagivg_kernels were about 50 percent husk-free.
All seeds were milled to about 20-40 mesh in a mechanical hammermill.
The resultant flour was extracted twice with 50 percent
aqueous acetone using 2 liters of 50 percent acetone per kg of flour.

The first extraction was for 6 hr and the filtrate was stored at 4 C.

The residue was then extracted an additional 18 hr, the filtrates
combined and the acetone removed jg_v§ggg, The extract was further
reduced to about l/20 of its original volume and stored at 4 C over-
night to permit removal of particulate material formed upon standing.
Centrifugation was at 9000 x g for l0 min in a Sorval RC-2 refrig-
erated centrifuge. The extract was then further reduced in volume
under vacuum to a brown sticky syrup about l/4OO of the original

volume and called "crude extract."

Chromatrographic Procedures

The resins used were purified according to the manufacturer's
instructions which includes alternate cycling through NaOH and HCl
for the Dowex 50 and DEAE-Sephadex resins. The mobile phase for the
Sephadex LH-20, Dowex 50 and Beckman PA 28 columns was 50 percent
2-pr0panol in water. The mobile phase for the DEAE-Sephadex column
was 50 percent ethanol in water. Free IAA was eluted from the latter
resin with a gradient from zero to five percent acetic acid in 50
percent aqueous ethanol.

Solvent systems for TLC were A solvent: methyl ethyl ketone,
ethyl acetate, water and absolute ethanol, 30:50:l0:l0; and G solvent:
chloroform, methanol, and water 85:l4:l (l7). Indole compounds were
detected on TL plates with Ehmann's spray reagent (l7) followed by
brief charring at 100 C. This reagent yields colors characteristic
of the particular indole. Paper chromatograms were run on Whatman

#l paper using A solvent and were sprayed with ninhydrin reagent(50).

For preparative TLC the sample was streaked on a 20 x 20 cm
TL plate and standards applied to one side. The chromatogram was
deveTOped in A solvent and after drying, UV fluorescent spots
outlined. The guide strip, containing the standard, was cut from
one side of the plate and the strip Sprayed with Ehmann's reagent.
The apprOpriate band(s) could then be scraped from the plate and
the sample eluted from the silica gel powder with 50 percent 2-
propanol.

Determination of the Amounts of
Free and Total IAA

 

The isotope dilution assay of Rittenberg and Foster (42)
as applied to IAA determinations in plants by Bandurski and Schulze
(5) was used for determinations of free and total IAA. A known

l4

amount of C-IAA of known specific activity was added to the

extract for determination of dilution. To assay total (free plus

ester) IAA the extract with the added 14

C-IAA was treated with l N
NaOH for l hr at room temperature, the extract acidified with H2504
to pH 2.5 and free plus alkali-liberated IAA extracted into CHCl3.
To assay free IAA only, the extract with added isotope was acidified
and extracted into CHCl3. Extracts were partitioned with an equal
volume of CHCl3 three times. The chloroform phase was dried over
anhydrous granular Na2504, evaporated to dryness jg_yagug, and
resu5pended in a minimal volume of 50 percent ethanol. The extract
was then chromatographed on a 3 ml bed volume DEAE-Sephadex column
as described earlier. Fractions containing radioactivity were

pooled, then further purified by preparative TLC.

The amount of IAA in the purified preparation was determined
by means of the Ehmann assay (40) and the radioactivity determined
as described above. Thus the specific activity was known. Using
the isotope dilution equation:

0
0
y=(—-l)x
cf

14

where Co the specific activity of the C-IAA added,

14

Cf the specific activity of the C-IAA recovered,

l4

x the amount of C-IAA added to the extract,

and y the amount of IAA present in the original extract, the

amount of IAA present in the original extract can be determined.

Spectrosggpy

Absorption spectra were determined with a Cary 15 recording

spectrophotometer.

Ammonolysis

 

The sample (usually 25 ul or 2 mg) was placed in a 2 ml
screw-cap vial and 50 ul of 58 percent NH4OH added. The reaction
mixture was heated at 45 C for 45 min, then cooled and evaporated
to dryness under N2. The residue was taken up in a small volume of
50 percent 2-pr0panol and assayed by TLC or GC. This procedure
yields approximately 50 percent free IAA and 50 percent indoleaceta-

mide from most esters.

Derivatization of Compounds
for GC and GC-MS

 

The sample was dried under N2 and then placed in a N2-
atmosphere chamber further dried with P205 (0. 0. Cohen). Remaining
water in the sample was removed by azeotropic evaporation with
added benzene. The dried sample was resuSpended in a small volume
of dimethylformamide and an equal amount of BSTFA added. The
stoppered reaction vial was heated at 45 C for 45 min. Samples

prepared in this way may be stored several days at room temperature.

e814;
Mass spectra were obtained using either a Hewlett-Packard

5985 GC-MS or a DuPont UP 102 GC-MS. Electron impact ionization

was at 70 eV. In the HP 5985 samples were introduced through a 1.2

m 2% OV-l column programmed from 220 to 280 C. In the DP 102 samples

were introduced through a 10 m OV-lOl capillary column programmed

from 130 to 300 C.

RESULTS

Quantitative Determination of Free
and Total IAA in O. sativa

 

 

The results of several determinations of the amounts of
free and total IAA in Q, sggiya_are shown in Table 1. There is
approximately 800 ug/kg of free IAA and 3000 ug/kg of total IAA for
a total of 2200 ug/kg of esterified IAA in seeds of Q, EEEili~
These figures are in reasonable agreement with previously published
values for commercially packaged brown rice, of 1700 ug/kg for free
IAA and 2700 ug/kg for esterified IAA (6).

Isolation of Esters of IAA
From 0. sativa

 

A flow diagram indicating the general procedure for purifica-
tion of the ester fraction is shown in Figure 1. About one kg of
flour prepared from dehusked grain was used for each preparation.

The first purification step was chromatography over Dowex 50.
The Na+ for of the resin was contained in a Buchner funnel with a
fritted disk bottom (6.5 x 5 cm) having a bed volume of about 100 ml.
The extract was layered on top of the resin and fractions eluted,
using a slight vacuum, with successive aliquots of 50 percent 2~
pr0panol. The separation obtained is shown in Figure 2. Ehmann-

positive material, at an Rf similar to that of authentic IAA-inositol,

lO

 

11

TABLE 1.--Resu1ts of the Quantitative Determination of Free and
Total IAA in Q, sativa.

 

Amount (us/k9)

 

 

 

 

 

 

Determination Variety Free Total Ester
#1 Calrose 830 n.d.1 --
#2 Calrose 392 2630 2238
#3 Calrose 1150 3390 2240

Average 791 3010 2239
#4 Comet 2 1
Brown Rice n.d. 2447 --
#5 Comet 2
Brown Rice 698 3108 2410
#6 Comet
Brown Rice 630 2232 1602
#7 Comet
Brown Rice 490 2126 1636
Average 606 2478 1883
#8 Comet 3
Brown Rice 183 495 312
#9 Comet 1
Brown Rice n.d. 539 ~-
1

n.d. Value was not determined.
2Values were measured using aliquots from an extract which
was reduced to only 1/50 of the original volume.

3These measurements were made using grain which had been

stored in its original containers for longer than one year.
The values obtained were not used in calculating average
values.

12

RICE GRAINS

 

GROUND
EXTRACTED WITH 50% ACETONE
FILTERED

 

 

FILTRAI§_ RESIDUE

REDUCED IN VOLUME
STORED IN COLD
FILTERED

 

 

FILTRATE RESIDUE
CENTRIFUGED

 

 

SUPERNATANT RESIDUE
(REDUCED
TO SYRUP

 

CRUDE EXTRACT

CHROMATOGRAPHED OVER DONEX SO
CHROMATOGRAPHED OVER BECKMAN PA 28
PREPARATIVE THIN-LAYER CHROMATOGRAPHY
CHROMATOGRAPHED OVER SEPHADEX LH-ZO

 

ESTER FRACTION

FIGURE 1.--Procedure for the Purification of IAA Esters.

 

FIGURE 2.--E1ution pattern from Dowex 50 of a
crude extract of Q, sativa.

A two ml aliquot from each fraction was dried
jg_vacuo, resuspended in 50 percent 2-propanol,
and applied to the thin-layer plate. Chromato-
grams were developed in A solvent and sprayed
with Ehmann's reagent. Volumes of fractions
1-4, 25 ml each; fractions 5-11, 100 ml each.

14

was observed in fractions 6-9. In general the esters elute between
300 and 600 ml and recovery is about 70 percent.

Fractions containing Ehmann-reactive substances were pooled,
evaporated to dryness jg_yggug, and resuspended in 50 percent 2-
propanol. The sample was then further fractionated on Beckman PA 28
resin in an HPLC system. The column was 0.9 x 17 cm and the sample
eluted with 50 percent 2-pr0panol using a pressure of 8.6 atm.
Fractions of 1.4 ml were collected, and 50 ul of every other fraction
assayed on TLC for IAA esters. With this colum esters generally
elute between 22 and 50 m1, and recovery of standard solutions of
indole compounds from this column is about 70 percent (11). Frac-
tions containing IAA esters were pooled, reduced in volume, and
upon chromatography, yielded the TLC profiles Shown in Figure 3.

The sample is still impure as colored material appears behind the
rice ester and UV-detectable flourescent material is still present.

The next purification step was preparative TLC. Losses with
this procedure are about 50 percent. Thus it is preferable to omit
the step and to further purify the produce on a 2 x 35 cm column of
Sephadex LH-20 having a bed volume of 84 m1 using 50 percent 2-
pr0panol as the eluent. Esters of IAA are eluted between 80 and 90
ml with less than 15 percent adsorptive loss.

TLC of the IAA ester preparation at this stage shows
Ehmann-positive material with an Rf and two-spot migration pattern

identical to that of authentic IAA-inositol isomers.

 

FIGURE 3.--IAA ester preparation from g. sativa after
chromatography over Dowex 50 and Beckman PA 28.

 

The rice extract was spotted on the left, the
IAA-inositol standard on the right.

l6

Ammonolysis

 

Ammonolysis of an IAA-inositol ester as described above
should yield three products; the free acid, IAA, the amide,
indoleacetamide, and the alcohol, mygfinositol. The products of
ammonolysis were chromatographed on TLC as shown in Figure 4. The
rice ester and authentic IAA-inositol Show Ehmann-positive material
at the same Rf before ammonolysis, and Ehmann-positive material at
Rf's corresponding to authentic IAA and indoleacetamide after
ammonolysis. In this system only indolylic compounds are detected.

That mygfinositol is also a product of ammonolysis was
demonstrated by GC. The TMS derivatives of the expected products
(IAA, indoleacetamide and m o-inositol) were chromatographed on a
1.8 m 1% OV-l column and their retention times at 170 C determined.
The ammonolysis products of authentic IAA-inositol and the putative
IAA-inositol from rice were derivatized and chromatographed under
the same conditions. Table 2 shows retention times for the standards
and the ammonolysis products.

The chromatographic pattern of the ammonolysis products from
the rice ester is identical to the pattern from authentic IAA-
inositol except that it lacks the peak at 0.9-1.0 min. Indoleaceta-
mide can be silylated once or twice at the amino nitrogen and the
second silylation depends upon the ratio of reagent to compound.
Thus the absence of the 0.9-1.0 min peak reflects the absence of
partially silylated indoleacetamide. Peaks at other retention times
correspond to those of the expected products of ammonolysis, IAA,

indoleaceamide, and myo-inositol.

l7

 

FIGURE 4.--Thin-layer chromatogram of the products
of ammonolysis.

Spots: l, IAA; 2, Indoleacetamide; 3, IAA-
inositol; 4, ammonolyzed IAA-inositol; 5, IAA;
6, Indoleacetamide; 7, rice extract; 8, ammono-
lyzed rice extract; 9, IAA; 10, Indoleacetamide.

18

TABLE 2.--Gas Chromatography of the TMS Derivatives of the Products
of Ammonolysis.

 

 

Compound Retention Time (min)
Indoleacetamide 0.9 - 2.1 -
Indoleacetic Acid - 1.5 - -
mygflnositol - - - 3.4

IAA-inositol, Ammonolyzed

Preparation 1 1.0 1.5 2.3 3.3

Preparation 2 1.0 1.4 2.3 3.3

Rice Ester, Ammonolyzed

Preparation 1 - 1.4 2.2 3.3

Preparation 2 - 1.4 2.2 3.3

 

19

Characterization of the 0. sativa
Ester by GC

 

The rice ester was further characterized by its retention
time during GC. The TMS derivatives of authentic IAA-inositol and
the putative IAA-inositol from rice were prepared and chromatographed
in two different systems as shown in Table 3.

In the first system a 1.2 m 2% OV-l column was used in a
Varian Series 2700 GC. The temperature was programmed from 220 to
280 C at 6°/min. In the second system a 1.8 m 1% OV-l column was
used in the HP 402 GC and the samples chromatographed isothermally
at 240 C.

Peaks from the rice ester at retention times of 1.9 and 2.3
min in the second system are contaminating material. The major
peaks in both systems coincide with those of the TMS derivative of
authentic IAA-inositol.

Characterization of the O. sativa
Ester by GC-MS

 

 

Final characterization of putative IAA-inositol from Q,
Mwas accomplished using GC-MS. The TMS derivatives of both
the rice ester and authentic IAA-inositol were prepared as described
above. The spectra in Figures 5a and 6a Show the 70 eV fragmentation
patterns for the axial and equatorial esters respectively, of the
TMS derivatives of authentic IAA-inositol.

The molecular ion, Mi, is at m/z 769. The major high mass
fragment ions characteristic of the axial ester occur at m/z 697 and

574, while high mass fragments characteristic of the equatorial

20

TABLE 3.--Gas Chromatography of the TMS Derivatives of IAA-Inositol
and of the Rice Ester.

 

Compound Retention Time (min)

 

I. 2% OV-l, 1.2 m, Programmed:
220-280 C at 6°/min

IAA-Inositol 5.9 7.3 8.7 9.5

Rice Ester 6.0 7.5 8.7 9.5

II. 1% OV-l, 1.8 m. Isothermal:

240 C
IAA-Inositol - - 2.6 3.8 4.6
Rice Ester 1.9 2.3 2.6 3.8 4.6

 

esters occur at m/z 679 and 607. Fragment ions characteristic of
the inositol moiety are seen at m/z 507 and 433 (53) while those
characteristic of the indole moiety occur at m/z 229 (base peak),
202, and 130. Other abundant peaks at m/z 147 and 73 correspond to
TMS fragments. The fragmentation patterns for the rice ester at
retention times corresponding to authentic IAA-inositol esters are

shown in Figures 5b and 6b and are essentially identical.

21

FIGURE 5.--Mass spectra obtained with the DP 102 GC-MS
of the axial ester of IAA-inositol.

a. Mass Spectrum of authentic hexakis-TMS-IAA-
m o-inositol, axial ester.

b. Mass spectrum of the TMS derivative of IAA-
m o-inositol from Q, sativa at a retention
time corresponding to that of the axial ester.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

IAA- INOSITOL
229
73 m
o
2 769
202
0 318
147
191 30
3 7
03130 343 O 574
I
I .I . i l L 697
I Y I fI I T I I I I
100 m 300 500 m m m
RICE ESTER
M?
229 769
u:
o
318
13
o\
202
305
343 507
1'47 19 462
130 574 697
m m 300 see 600 m sea

 

 

 

 

23

FIGURE 6.--Mass spectra obtained with the DP 102 GC—MS
of the equatorial ester of IAA-inositol.

a. Mass spectrum of authentic hexakis-TMS-IAA-
m o-inositol, equatorial ester.

b. Mass spectrum of the TMS derivative of IAA-
m o-inositol from Q, sativa at a retention
time corresponding to that of the equatorial
ester.

 

24

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1AA - INOSITOL
229
LIJ
Z 73
<1
0
Z
I)
<
202 x 4
\° 2‘
° 318 / M
769
679
343 433452 507 J
5A."...fi1Ln4—nq-lnr5fi‘fiflnn.ranun .L
m 500 600 m m
"V1
RICE ESTER
229
1.1.1
0
Z
d
0
g x 4
m 73 /
4
3
202 318
M‘!‘
147 .9. a 303 769
343 462 507
,03'30 ‘33 607 679
100 200 300 m see 690 m 800

 

"V1

 

 

25

Isolation and Partial Characterzation
of Tryptophan from 0. sativa

 

 

Chromatography of a crude extract of Q, satjyg_over Dowex 50
gives an Ehmann-positive material with an Rf on TLC different from
that of IAA-inositol. This material is apparent in fractions 7-11
of Figure 2. Since it occurs in large amounts it was of interest
to see if it contained IAA. Ammonolysis does not yield detectable
IAA or indoleacetamide, so it is not an ester. Comparison of its
migration pattern on TLC with other known compounds showed that it
had an Rf corresponding to that of tryptophan (Figure 7). In
addition, the product is ninhydrin-positive.

When this rice product was assayed with Ehmann's reagent,
as Shown in Figure 8, the Spectrum from 800 to 400 nm was like that
obtained with authentic tryptophan. Both Show a broad peak with a
maximum at 600 nm, unlike a profile of IAA which has a maximum at
615 nm.

UV Spectra for L-tryptophan and the putative tryptophan
from rice, after purification over Dowex 50 and Beckman PA 28 are
shown in Figure 9. The absorption spectrum of the rice product is
identical to the absorption Spectrum of tryptophan.

An attempt was made to examine the MS fragmentation pattern
of the TMS derivative of the rice product using both the HP 5985
GC-MS and the DP 102 GC-MS. Published spectra of silylated
tryptophan (20,31) show that M? is at 420 but M - 15 at m/z 405 is
the highest mass actually observed, and that m/z 130 is only a minor

peak. Base peak for both authentic L-tryptophan and the rice

26

 

FIGURE 7.--Thin-layer chromatogram of indoleacetylaspartate,
tryptophan, tryptophan from Q, sativa, and tryptamine,
developed in G. solvent.

Spots: l, IndoleacetylaSpartate; 2, authentic L-
tryptophan; 3, tryptophan from Q, sativa; 4, trypta-
mine.

ABSORBANCE

27

 

 

 

 

I I I T U I I
0.7 -
0.6 -
0.5 r-
0.4 -

C
B

0.3 - A
0.2 -
0.1 - A: L-TRYPTOPHAN. 5 pg

8 1 L-TRYPTOPHAN, 10 pg
0.0 - C1 SAMPLE

l L L l l L 1
450 500 550 600 650 700 750

WAVELENGTH (nm)

FIGURE 8.--Visib1e light spectra of authentic
L-tryptophan and tryptophan from
Q, sativa after Ehmann assay.

28

 

 

 

 

 

 

 

 

 

 

T I I
0-8 t A: L-T'RYPTOPHAN
a: SAMPLE
0.7 . 11 .
0.6 '- cl
0.5 '- cl
3 0.4 - -
Z
<
§§<DEIP .
O
“ a A
0.2 - .
0.1 1' . 4
0.0 - -
1 l l
230 300 350

WAVELENGTH (nun)

FIGURE 9.--U1traviolet Spectra of authentic L-tryptophan
and tryptOphan from Q, sativa.

29

product in this study (data not shown) is m/z 130, but that particu-
lar fragment would be expected for any indolylic compound. Neverthe-
less, on the basis of the evidence presented above, we tentatively

conclude that this indolylic compound is tryptophan.

DISCUSSION

Quantitative Determinations of IAA

 

In the quantitative determinations of free and total IAA
there is considerable variation in the values obtained using the
isotope dilution assay (see Table l), but the value for esters of
IAA is relatively constant from one preparation to another. Several
explanations for the apparent variability may be given; I would like
to discuss two of them. Other discussions of possible errors
resulting from the use of the isotope dilution assay are presented
by Rittenberg and Foster (42) and by Bandurski and Schulze (6).

First, to calculate the total amount of IAA present in the
original extract (y) requires that the amount in the added labeled
IAA (x) be accurately determined. Furthermore the amount of IAA in
the isolated fraction (x + y) must be known precisely; this becomes
very difficult because the amounts involved are very small. Since
there is a very small amount of free IAA present in a plant extract
and since recovery of that may be only 25 to 30 percent, errors in
measurement can be magnified.

In the experiments reported here the Ehmann colorometric
assay was used to determine the amounts of IAA. This assay, like
the Ehrlich assay, depends primarily on the chemical reaction

between an indolylic compound and para-dimethylaminobenzaldehyde,

30

31

and the subsequent oxidation of the product in the presence of
strong acid (l5,l7). Upon dilution of the reaction mixture the
absorbance of the product at 615 nm is proportional to its concentra-
tion and is linear over a concentration range from 0.1 to 50 ug of
IAA (Ehmann, unpublished results). The value obtained for the
absorbance of the product at 6l5 nm can be treated in two different
ways to determine the actual amount of IAA present in the sample
being assayed. The first relies on data accumulated in this
laboratory over a period of several years, that is, multiplying

the absorbance at 615 nm by 9.2. The product gives the amount of
IAA in ug for the particular sample assayed. Obviously the method
depends on accurate measurement of small volumes of sample, on the
consistent response of the Spectrophotometer, on the reagent having
the same composition each time, and on the reaction conditions
being the same each time.

In preference to assuming absolute absorbancy, a calibrating
reaction can be run simultaneously on several samples containing
known amounts of IAA at the same time as the assay of the unknown
is being performed. A standard response curve can then be constructed
and the amount of IAA in the unknown sample determined by comparison
of its absorbance with those on the standard curve. This eliminates
variation due to different batches of Ehmann's reagent. It does
depend on having a standard solution of IAA of known concentration;
this was obtained by determining the absorbance of the standard at
280 nm and calculating the amount present using the Beer-Lambert

law:

32

A = ebc
where A = absorbance,
c = molar absorptivity (assumed to be 6060),
b = path length, (cm)
and c = concentration(moles/l).

A second explanation for the variability derives from the

l4 14

use of C—IAA as the internal standard. C-IAA is probably the

ideal internal standard for measuring free IAA because losses of

endogenous free IAA and added 14

14

C-IAA will be the same during puri-

fication. Using C-IAA as the internal standard for measuring total

IAA is not as satisfactory. For one thing, losses of esters or

amides of IAA which occur prior to alkaline hydrolysis cannot be

14MM is more labile than is IAA in

14

accounted for. Second, free
a conjugate; oxidative destruction of free IAA and free C-IAA
prior to alkaline hydrolysis of IAA conjugates would lead to an
overestimation of the total IAA. If for some reason hydrolysis were
incomplete, the amount of total IAA would be underestimated. In
experiments reported here, the conditions are sufficient for com-
plete hydrolysis of IAA esters. 14C-IAA-inositol could be used as
an internal standard for measuring total IAA, but the values obtained
would not necessarily apply to the entire ester fraction.

Consistent handling of plant extracts is important to reduce
losses of IAA and IAA conjugates during measurements. In most of

the determinations reported in Table l the crude extract was a small

volume of thick syrup, whereas in determinations #4 and #5 aliquots

33

from a large volume of extract reduced to l/50 of the original

volume were used. It seems very probable that the total recovery

of free and total IAA depends on the degree to which an extract is
reduced in volume. At least it has been noted that extensive concen-
tration of sweet corn extracts results in the loss of low molecular
weight IAA esters (52). Presumably the IAA complexes with polymeric
materials in the extract so it is not released by l N base hydrolysis
or it precipitates with high molecular weight esters.

Treatment of the sample during extraction, exclusive of
volume reduction, may also account for IAA losses. Mann and Jaworski
(35) reported losses of up to 52 percent during extraction of IAA
with ethanol. Previous work in this laboratory (52) indicates that
with pr0per handling, losses during extraction are about 20 percent.
The age of the grain also seems to be important. ImprOperly stored
or old grain seems to contain less total IAA (see Table l).

Another possible source of error is incorrect determination

of the specific activity of ‘4

C-IAA added. If the recovered sample
is not well purified, other compounds absorbing at 6l5 nm may inter-
fere with the Ehmann assay. Usually this can be detected by showing
that TLC plates of the isolated IAA have a single spot producing
color with Ehmann reagent, and by comparing the absorption spectra
of sample and standard after the Ehmann assay.

The value reported here for the free IAA content of Q, sativa

is half that reported by Bandurski and Schulze (6), but the value for
esters of IAA, 2200 pg/kg, is close to their published value of

34

2700 ug/kg. IAA released from conjugates by 7 N hydrolysis (ester
plus amide conjugates) was not determined.

Early studies of plant auxins were concerned with both
isolating and characterizing the auxins and with attempts to measure
the amounts present, but the fact that very small amounts occur in
plant material meant that yields of auxin were very small. (Consider
the alternate route of Haagen-Smit et al. (24) who extracted l00 kg
of fresh corn kernels which yielded 3700 ml of thick syrup from
which they were able to purify l.20 g of IAA.) Nevertheless several
early workers were able to purify IAA and characterize it by
chemical criteria: crystal formation, melting point analyses (23),
solubility properties (3), and infrared spectroscopy (8).

Not all workers were careful to characterize the compounds
isolated. Very often the criterion for the presence of auxin was
its activity by bioassay or, occasionally, by its migration in
paper chromatography. In spite of these limitations some estimates
are remarkably close to those obtained using assays such as isotope
dilution. Avery et al. (3) examined several plant materials. Their
estimate of water-extractable IAA (which may be considered free IAA)
from rice was 800 ug/kg while alkali extraction yielded 2500 ug/kg.
Sircar and Das (47) also examined rice, and their alkali extraction
procedure yielded 5000 ug/kg of IAA, although in a later paper (46),
using a different bioassay, Sircar found 50 ug/g of endosperm
which is l0 times the earlier value. Sircar and Chakravorty (46)

attempted to determine the location of IAA in the rice grain and to

35

measure the changes in distribution during develOpment and germina-
tion. They noted that most of the IAA was present in the endosperm
of the dry seed, while the embryo and scutellum contained less than
one percent. During germination however, the amount of IAA in the
endosperm declined (47), but Sircar reports that not all the IAA
that disappeared was recovered from the embryo (46). Perhaps Sircar
appreciated some of the problems involved in accurately measuring
the amount of a material which is not also chemically characterized
when he wrote, "Such crude auxin extract in water from the endOSperm
and embryo used in their experiments [46,47] were obviously of a
mixture of IAA, GA and other naturally occurring growth substances.
It is now pertinent to reinvestigate the problem of the intracellular
distribution of different growth substances in the rice seed and the

role they play in controlling germination" (45)-

Isolation and Purification of Esters

The procedure described above for isolation and purification
of an ester fraction is only slightly modified from the procedure
described by Cohen and Bandurski (ll) for purification of the B
esters from sweet corn. Applying the method described here to Zea,
one can detect all 5 of the low molecular weight esters found by
Ueda et al. (54). This same procedure failed to detect any low
molecular weight IAA esters in A, sgtjyg,

A purified extract prepared from l kg of dehusked rice
kernels yields approximately 200 ug IAA-inositol. This value has not

been precisely measured, but is based on estimates from TLC and

36

corrected for recoveries reported for the various chromatographic
steps. Thus, IAA-inositol appears to be the only low molecular
weight ester, but accounts for less than l0 percent of the total
esterified IAA present.

In early experiments two other procedures for the isolation
of IAA esters were tried using Comet Brown Rice. The first was a
standard method for fractionation (54) which includes partitioning
the crude extract at a neutral pH against l-butanol. This can be
followed by a second partitioning against l-butanol at a lower pH.
The remaining aqueous phase is centrifuged which results, in the
case of Q, sativa, in three zones. All were assayed for Ehmann-
positive material and for IAA and indoleacetamide formation after
ammonolysis. The lowest zone, a thick amber syrup, yielded IAA and
indoleacetamide, and an assay of this fraction for total IAA indicated
it contained a high pr0portion of the esters present.

To estimate the size of the esters contained in this fraction,
the fraction was dialyzed against 50 percent 2-propanol. After l8
hr dialysis at 4 C the solvent outside the dialysis sack was removed
and evaporated to dryness jn_vggug, TLC confirmed the presence of
low molecular weight ester, so the fraction was further purified by
chromatography over Sephadex LH-20 followed by preparative TLC.
Ammonolysis indicated that the isolated product could be an IAA
ester, but too little product was obtained to permit further charac-
terization. TLC in A solvent (methyl ethyl ketone, ethyl acetate,

ethanol, and water) of the material remaining inside the dialysis

37

sack suggested the presence of an Ehmann-positive material which
remained at the origin. TLC of this product in a solvent containing
2-pr0panol, water and NH40H, 7:2:l, yielded Ehmann-positive material
with a high Rf. These observations strongly suggest that the
dialysate contained high molecular weight, reactive complexes of
IAA, but this fraction was not further characterized.

Another approach to isolation of IAA esters was used which
attempted to take advantage of the fact that a crude extract of rice
is innately "clean" compared to extracts of other seeds such as Egg_
or BXEDE: Some fractionation of the crude extract was obtained by
chromatography over Sephadex LH-20 with 50 percent 2-pr0panol as
the mobile phase. Early fractions contained much carbohydrate but
at 20 to 30 ml an Ehmann-positivenmterialeluted. 0n TLC with A
solvent the spot did not migrate, but ammonolysis indicated it was
an ester-like complex of IAA. After further purification of this
fraction using TLC, an isotOpe dilution assay indicated it contained
about 5 percent of the total esterified IAA, but recoveries were too
poor to allow further characterization. Because no other IAA com-
plexes were eluted from the Sephadex LH-20 column, and only a small
part of the bound IAA had been accounted for, it seemed possible
esters were being retained. Elution from the column at pH 9.5 with
buffered borate (40 mM) in 50 percent 2-propanol did not yield any
additional IAA. Passage of a similar crude extract Spiked with
14C-IAA over Sephadex G-l0 with three different mobile phases, water,

50 percent aqueous acetone,and 0.l N NaOH in 50 percent aqueous

38

acetone failed to elute any unusual amounts of Ehmann-positive

MC-IAA

material, or, in the case of the final solvent, to dilute the
significantly.

These experiments suggest that a large part of the esterified
IAA in Q, satjya_has a high molecular weight. It may be esterified
with polysaccharide as in the A fraction of sweet corn (4l) or with
gluc0protein as in Avggg_(40). The failure to recover IAA-containing

material from either the Sephadex LH-20 or G-lO column remains to be

explained.

Esters of IAA in Husks of 0. sativa

Since dehusking of rice grains is laborious it was important
to see if IAA esters could be extracted from the husks; if not, then
perhaps unhusked grain could be used. The husk preparation obtained
was not entirely free of small pieces of rice endosperm. The amount
of IAA-inositol detected after chromatrography over Dowex 50 was less
than 5 ug/kg and was probably the result of contamination of husks
with pieces of grain. The husks do contain free trypt0phan and
some phenolic compounds (not characterized) which might interfere
with ester purification from the grain, or with any quantitative
studies; thus it appears that most of the husks should be removed
for studies of the kernels.

Mass Spectrometry and Interpretation
of Spectra

 

 

The spectra of putative IAA-inositol from rice (Figures 5b

and 6b) are identical to those of authentic IAA-inositol (Figures

39

5a and 6a) as obtained from the DP l02 GC-MS. The differences
between mass spectra presented here and published spectra (20,53)
arise from differences in derivatization procedures and instruments
used. This is illustrated by comparing published spectra of IAA-
inositol, those obtained with the DP l02 GC-MS (Figures 5 and 6),
and those obtained with the HP 5985 GC-MS (Table 4).

Table 4 presents mass abundancies of higher mass fragments
at three different retention times for authentic IAA-inositol and
for the rice product. Table 4 shows that with the HP 5985 GC-MS
the higher mass fragments characteristic of IAA-inositol analyzed
in different types of mass spectrometers are present, but that only
some of the fragments are equally abundant. In the case of the
rice product, M? at m/z 769 is observed for only one isomer of IAA-
inositol, the axial ester. In general the most abundant fragments
reported by Ehmann et al. (20),obtained with a magnetic sector
GC-MS, are also the most abundant observed with the HP 5985 GC-MS.

Since the instruments differ in the way ions are separated,
the information gained from the spectra differs. The HP 5985 GC-MS
is a quadrupole MS and the resolution possible at higher masses
reduces its sensitivity in the higher mass range. This means that
a large amount of sample must be used so that a significant propor—
tion of it which survives the ionization process also survives the
separation process. The result is that M? is hard to detect, but
the lower mass fragments are abundant. The DP 102 GC-MS is a
magnetic sector instrument, but it is unusual in that the accelerat-

ing voltage, not the magnetic sector, is scanned. Varying the

40

TABLE 4.--Relative Abundancies of Ions in the Mass Spectra of the
TMS Derivatives of Authentic IAA-Inositol and IAA-Inositol
Purified from Q, sativa.‘,2

 

Retention Time

 

 

Compound 6.9 min 7.6 min 8.l min
Mass Abundance Abundance Abundance
(amu) (%) (%) (%)
Authentic

IAA-Inositol 769 0.3 (l5.0) 0.l (ll.2) 0.1
697 .l (0.8)
679 0.4 (0.9)
706 0.l (0.3)
507 0.l (2.l) 0.2 (l.4) 0.1
462 0.l (0.8) 0.l (0.4)
433 0.3 (2.3) 0.7 (l.7) 0.3
4l7 0.2 (l.4) 0.5 (l.0) 0.3
394 0.l
391 0.2
361 0.1 0.l (0.5)
343 l.4 (3.3) 2.4 (2.0) 0.9
3l8 3.0 (8.3) 10.5 (7.5) 1.9

IAA—Inositol
from Q, sativa

769 0.4

433 0.3 0.1

417 0.7 0.2

343 1.9 1.3 1.0
318 5.9 3.1 3.4

 

1These spectra were obtained using the HP 5985 GC-MS.

2The numbers in parentheses are from Ehmann et al. (20).

4l

accelerating voltage results in the separation of ions on the basis
of momentum and has the fortunate effect, in the case of IAA-
inositol, that the molecular ion and other higher mass ions are
more abundant.

It is interesting to note that the sample was introduced
into the DP 102 GC-MS by chromatography over an 0V-l01 capillary
column l0 m in length. Two GC peaks were observed and although
the spectra for these 2 peaks are excellent, no other peaks were
observed (data not shown); using packed columns, resolution of the
ester into its various isomers is good and repeatable (18).

Mechanisms accounting for the major fragments from axial
and equatorial esters of IAA-inositol have been disucssed by Ehmann
and Bandurski (l9). M? loses 72 amu to give a peak at m/z 697
(Figure 5) and Ehmann and Bandurski (19) demonstrated that this
represents the loss of the TMS group from the indole N and the
transfer of a hydrogen atom from a methyl group of the N-TMS to
the indole ring. Although these authors found m/z 697 characteristic
of both axial and equatorial esters I found it characteristic of
one isomer and not the other. The same isomer has a characteristic
peak at m/z 574. This fragment is reported by Ehmann and Bandurski
(l9) and by Ueda and Bandurski (53), and is attributed to the loss
of two TMSOH groups and one CH3 group (Mt-l5-90-90), all presumably
from the inositol ring. The fragmentation patterns suggest that
with the DP l02 GC-MS the spectrum obtained from a peak at one

retention time represents the axial isomer, and that the spectrum

42

obtained from a peak at a later retention time represents the
equatorial isomers.

The equatorial ester seems to be characerized by loss of
90 amu (Figure 6) which would represent a TMSOH group leaving which
was also reported by Ehmann and Bandurski (l9). They report that
this interpretation is supported by a metastable peak at m/z 600
with the subsequent formation of a fragment at m/z 607. The ion at
m/z 607 represents the loss of a -CH2(CH3)ZSi fragment. I do not
observe the m/z 36l reported by these authors.

The ion at m/z 462 might correspond to that ion noted by
Ueda and Bandurski (53) at m/z 390. Their M? was not silylated at
the indole N, consequently ions they report which contain the indole
ring have masses 72 amu less than those reported here. They
interpret the ion at m/z 390 as resulting from the loss of a C3
unit from the inositol ring. Other fragment ions characteristic
of either the indole ring or the inositol moiety have been reported
before (l4,44,56) and are summarized by Ueda and Bandurski (53).

Since the spectra of authentic and isolated IAA-mygfinositol
in the same instrument are nearly identical, and since the spectra
presented here correspond closely with previously published spectra,

it is concluded that the isolated rice produce is IAA-_y97inositol.

Free Tryptophan in 0. sativa

 

A crude estimate of the amount of free tryptophan in g,
sativa suggests that after chromatography over Dowex 50 and Beckman

PA28 there is 500-1000 ug/kg present. Sircar and Dastidar (48) were

43

unable to detect tryptophan in rice grains until 48 hr after
germination. Sircar has (45) suggested that tryptophan might be
synthesized from serine and indole, the indole being produced by

the embryo as it used IAA. Other workers have found trypt0phan
present as the free amino acid in rice at various stages. Cagampang
et al. (10) report the very high value of 0.0l - 0.03 mg/g dry
weight free tryptOphan present in different lines of Q, sativa_
which vary in protein content. Lee and Kim (30) report the presence
of tryptophan in both the embryo and endosperm.

Several workers have been interested in the role of free
tryptOphan or in the relationship between free tryptOphan and IAA
during growth of the rice plant. Sircar and Lahiri (49) found that
tryptophan could substitute for IAA in a growth medium used for the
culture of rice embryos. Lee and Kim (30) also cultured rice
embryos on tryptOphan-containing medium, but found that in these
embryos the concentration of serotonin increased, and that S-hdroxy-
tryptOphan could be detected. The conversion of tryptophan to IAA
by a crude extract of immature rice grains has been reported by
Murakami and Hayashi (36) but they concluded that only 0.5 percent

of the IAA arises from tryptOphan in this way.

CONCLUSIONS

IAA- o-inositol can be isolated from extracts of Q, satjya_
kernels. It can be purified by chromatography over Dowex 50, Beckman
PA 28, and Sephadex LH-20. The ester nature of the linkage between
IAA and mygfinositol is demonstrated by the formation of IAA,
indoleacetamide and mygfinositol upon ammonolysis. These products
were identified by TLC and GC. Identification of the ester was
made by comparing its Rf on TLC, its retention time in BC, and its
fragmentation pattern in GC-MS as compared to authentic chemically
synthesized IAA-inositol.

A quantitative determination of the amount of IAA-inositol

14C-IAA-inositol but has not

in g, sativa_would be possible using
yet been done. Based on recoveries from various steps in purifica-
tion, I estimate there is 200 ug/kg of this ester. The amounts at
each step are estimated by comparison of spots on TLC with a
standard of known concentration. This means that approximately
l0 percent of the total ester IAA in Q, sativa is IAA-inositol.
Compared to sweet corn which contains 6000 picomoles of IAA-inositol
per seed, rice has only 12-l3 picomoles per seed.

The work reported here suggests questions other than how

much IAA-inositol is present. One question is what is the nature

of the remaining, apparently high molecular weight, esters of IAA;

44

45

are they like the IAA-gluc0protein fraction found in Avena (40),

or like the IAA-cellulosic-glucan of Zea (41)? Another question
concerns the large amounts of free tryptOphan present in the seed.
There are numerous reports in the literature concerning the conver-
sion of tryptophan to IAA in plant tissues (36,43,55). However, in
corn seedlings, the conversion rate of tryptophan to IAA is insuf-
ficient to meet the demand for IAA in the seedling (25). The ques-
tion is an Open one and might be of considerable importance in
understanding rice physiology.

There is a question of the importance of finding IAA-inositol
in rice. Is there any significance attached to mygyinositol being
bound to IAA? Presumably esterification between IAA and any number
of low molecular weight sugars or sugar alcohols is possible, and
does occur in sweet corn, yet in sweet corn IAA-inositol esters
predominate. In terms of total sugars inositol is a very minor
component in any seeds assayed (9,27,28,57). Most inositol is
present as phytic acid in seeds (2), while in growing plants
inositol is one compound in the synthetic route to cell wall com-
ponents (32,33). Studies of the inositol components of seeds have
shown that seeds vary in the amounts of free and total inositol
present. A summary of some data is listed in Table 5. The fact
that the Zga_contains relatively large amounts of inositol compared
to Dry a and Avgga_might be thought to correlate with the amounts
of IAA-inositol esters; however, while Avgga_contains inositol in
amounts comparable to 93153, I have been unable to demonstrate IAA-

inositol in Avena. As described earlier, several physiological

46

TABLE 5.--Free and Total Inositol in Seeds.

 

Total Inositol Free Inositol

 

Corn (hybrid strains) 150-200 70-120 26
Oats 90 3.0 l3
Rice - dry seed 40 0.9 28
30 days after
flowering 240 9.0 2
aleurone 9 ug/g 0.25 ug/g 5l
particles aleurone aleurone
particles particles

 

functions of IAA-inositol in Zgg_have been demonstrated; besides
its role in IAA transport it also serves as a seed auxin-precursor,
and esterification of IAA to m o-inositol protects the indole
moiety from oxidation by peroxidase. This study has not been
concerned with the physiology of IAA esters in rice, so their role
cannot be commented upon.

This is the first demonstration of IAA-inositol in a plant
other than gga_and its close relatives, and its occurrence in two
such widely separated graminaceous plants is very striking and of
possible importance. This is also the first report of the chemical

characterization of any IAA compound from kernels of Q, sativa.

REFERENCES

47

10.

11.

12.

REFERENCES

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Avery, G.S., Jr., J. Berger, and B. Shalucha. l94l. The Total
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Bandurski, R.S. 1979. Homeostatic Control of Concentrations
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