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Levels of IAA during Kernel Development and the Use of in Vitro Endosperm Systems for
Studying IAA Biosynthesis
was synthesized within the seed. Zea mays endosperm sus- required for sustained growth. Cultures were grown in the
pension cultures have been initiated and grown for many dark at 29OC on a rotary shaker at 120 rpm. Two I-L stock
years without the need for hormone supplementation (Shan- cultures grown for 7 d after subculturing servecl as a source
non, 1982). The in vitro culture of Z. mays kemels also of inociulum for the experimental flasks. Each stcck flask was
proceeds on hormone-free media (Gegenbach, 1977). Since subcultured into a set of six 250-mL flasks, each containing
these two cell culture systems grow without the need for 80 mL of medium. Every 2 d after subculture orte flask from
hormone supplementation, it is reasonable to conclude that each set was harvested, fresh weight was determined, and
these systems can make a11 of the hormones required for the tissue was lyophilized for later analysis. Z. mays kemels
sustained growth. (hybrid 873 X LH51) grown in vitro were provided by Dr.
In this study we describe a time course for the biosynthesis Fred Below of University of Illinois. The kemels were grown
of free and ester IAA in the developing endosperm of Z. on solid medium containing Murashige-Skoog salts, 15%SUC,
mays. These data demonstrate that free IAA is esterified as 2 g/L ,4sn, and 0.4 mg/L thiamine HCI. No hormones or
rapidly as it is synthesized in the kemel. Free IAA levels do aromatic amino acid were added to the media. I<emels were
increase, but the amounts of free IAA are small and can be harvested 20 and 32 DAP and lyophilized.
accounted for by the relationship between the free energy
changes of the first two conjugation reactions (Kesy and Feeding of Labeled Precursors
Bandurski, 1990). In much earlier studies using less exacting
analytical methods, Avery et al., (1942a), Teas and Newton Ears of com were surface sterilized for 10 nun in a 1:lO
(1951), and Corcuera (1967) for maize and Hatcher for rye dilutiori of commercial bleach containing 5% NaOCl and
(1943) described the time course for IAA accumulation. Brief then rirised four times with sterile water. The entlosperm was
reports of conclusions of this work have appeared previously excised from the kemels, and two endosperm were placed in
(Jensen and Bandurski, 1991; Jensen et al., 1992). sterile ~nicrofugetubes and gently homogenized with a glass
A second objective of this study was to determine whether rod. Additional endosperm were frozen at -8OOC for later
endosperm suspension cultures and cultured kemels produce analysiis to determine the stability of the system to freezing.
IAA de novo. We examined the feasibility of using homoge- Altematively, many endosperm were placed i11 a sterilized
nized Z. mays endosperm to study IAA biosynthesis. To test mortar, homogenized, and then transferred to sterile micro-
this system, ['4C]anthranilicacid was used as a putative early fuge tubes in amounts approximately equal to two endo-
precursor, and we demonstrated the attractiveness of the sperm. To each tube approximately 250,000 dpin (8.6 nmol)
maize endosperm as an experimental system for studies of of [14C:lanthranilicacid was added. Control siimples were
IAA biosynthesis. boiled for 5 min and cooled prior to the adclition of the
radiolabel. The samples were allowed to incubate for O, 4,
12, and 24 h. To stop the reactions an equal volume of 2 N
MATERIALS AND METHODS NaOH was added to inactivate the enzymes arld hydrolyze
Plant Material the IAA conjugates. The samples were then analyzed i"e-
diately or frozen with liquid nitrogen and stored at -8OOC
For the developmental study, experiments were conducted for Iater analysis.
on sweet com (Zea mays L., cv Silver Queen) grown under
field conditions. Ear shoots were bagged prior to silk emer- Sample Extraction
gente and then sib pollinated when the silks emerged. In an
attempt to compensate for variabihty due to environmental For the determination of free and total IAA in the devel-
conditions, two groups of ears were pollinated on different opmental study, 15 to 90 fresh kemels, depentiing on age,
dates. For IAA determinations, two ears from each pollination were extracted with aqueous acetone (final concentration
group were harvested every 5 d from O until 45 DAP. 70%, v/v) after being frozen with liquid nitrogen and ground
Additional ears were harvested 58 DAP and allowed to dry to a fine powder with a mortar and pestle. An intemal
at room temperature. Free and total IAA determinations were standard of [l3C~]IAA(100 pglsample) and approximately
made on fresh tissue. Each sample consisted of kemels from 80,000 cpm of [3H]IAA, to facilitate peak detxtion, were
one ear for a total of four samples per age group. To examine added to the samples. The samples were allowed to extract
the conversion of [14C]anthranilate to IAA, additional for 18 h at 4OC and then centrifuged for 5 min i11 a benchtop
ears were harvested between 22 and 30 DAP and used centrifuge at 1300g; the supematant liquid was divided for
immediately. free and total IAA analysis.
Suspension cultures of endosperm cells derived from the For the detennination of free IAA from the lyophilized
Z. mays inbred line A636 were kindly provided by Dr. Jack endosperm cells, 5% of each sample, based on dry weight,
Shannon of Pennsylvania State University. This cell line was was extacted with 70% acetone in vacuo for 2 h at room
established in 1986 from kemels 10 DAP. A complete de- temperature. Intemal standards were added as above. After
scription of the initiation and characterization of maize en- 2 h the samples were centrifuged as above, and the super-
dosperm suspension cultures has been previously reported natant was reduced in volume to an aqueou:, phase and
(Shannon, 1982; Felker et al., 1989). The cells were grown diluted with 2 mL of water in preparation for column
on liquid media, pH 5.6, containing Murashige-Skoog salts chromatography.
(Murashige and Skoog, 1962), 3% SUC,2 g/L Asn, and 0.4 For the determination of total IAA from the lyophilized
mg/L thiamine HC1 (Shannon, 1982). No auxin addition is tissue, an additional5% of the sample, plus interna1standard,
was extracted and simultaneously hydrolyzed with 1N NaOH monitored and peak areas measured to determine the
in vamo for 2 h at room temperature. The sample was then "C/"C ratio. Additional samples were scanned from m/z 50
acidified to pH 2.5 with 1 N HC1 in preparation for column to 350 to check for cleanliness.
chromatography .
RESULTS
IAA Purification
IAA Levels during Kernel Development
Free and total IAA were purified using a modification of a Developing kemels of Z. mays contain levels of IAA severa1
method developed by Chen et al. (1988). Quantification was orders of magnitude greater than reported for vegetative
accomplished through the use of stable isotope-labeled IAA tissues (Bandurski and Schulze, 1977). For the first 10 DAP
as an intemal standard (Magnus et al., 1980; Cohen et al., IAA levels remain constant at approximately 0.018 pg ker-
1986). The extract was applied to a DEAE-Sephadex A-25 nel-' (445 ng g-' fresh weight). Beginning 10 to 15 DAP,
column (acetate form, 1.5mL bed volume; Pharmacia) packed there is nearly a 5000-fold increase in the amount of total
in a 3-mL syringe fitted with a polypropylene frit. Column IAA, reaching a maximum of 92 pg kemel-' (160,000 ng g-'
washing and elution were as described by Chen et al. (1988). fresh weight) about 45 DAP and then decreasing by approx-
After elution, the samples were dried in vacuo, taken up in imately one-half in the dry seed (Fig. 1A). The change in IAA
solvent, and chromatographed using an HPLC (LDC/Milton levels parallels the tissue fresh weight (Fig. lB), indicating
Roy model CM 4000 pump with a model 3000 Spectro that the tissue monitors concentration and not amount per
monitor variable UV detector set at 280 nm) equipped with kemel. The increase of 92 pg kemel-' between 10 and 45
a Cls column (5-pm ODS, 25 X 4.6 cm, Val-U-Pak; Regis, DAP translates to an average IAA accumulation rate of 190
Morton Grove, IL) using 20% acetonitrile containing 1% ng g-' fresh weight h-'. This value may or may not equal the
acetic acid as the mobile phase. The 3-mL fraction at the kemel's IAA biosynthetic rate because it is not known
retention time of IAA was checked for radioactivityand dried whether a11 of the IAA made is conjugated and stored,
in vacuo. The sample was then methylated with diazome- whether there are losses during development, or whether
thane and subjected to a second C18HPLC step using 45% some IAA is imported into the kemel from the mother plant.
acetonitrile as the mobile phase. The free IAA fraction was Plotting free and total IAA on a per gram dry weight basis
then dried in vacuo and dissolved in acetonitrile for GC-MS indicates that IAA levels increase at a rate independent of
analysis. the change in dry weight until d 20 and then decline (Fig.
Altematively, extracts from the lyophilized tissues were
loaded by means of a vacuum manifold onto a preconditioned
10- X 10-mm semiprep guard column (Upchurch Scientific, 1 O0
Oak Harbor, WA) containing C18pellicular packing (What- 90
man, Clifton, NJ). After the precolumn was washed with 10 n
- 80
mL of water, it was inserted in place of the injector loop on Q)
E 70
the HPLC and further purified as above.
For the determination of total IAA in the developing en- 4 60
400
1000 1
,I
T T
A Total IAA 350 p
c
+ A Free IAA o)
3 800 300 E
o)
? 250
o
600
2
2oo E
400 150-
v
100 z
Q 200
9 50 6
O O
600 p
2 200 o)
3 500 E
>
0)
L Y Days After Pollination
150 400
o)
L
w- Figure 3. The levels of free IAA per kernel during development of
E
:3
lo0
50
300 v
200 z
L
Z. may!; kernels in situ. Vertical bars indicate the SE of four replicate
samples.
a 100 !g
-
6 L
O O
LL growth, the time of maximum free IAA cannot be conclu-
O 10 20 30 40 50
I /
60 sively determined, although the data suggest a maximum of
approximately 4 d after subculture. There is clearly a decrease
Days After Pollination
in free IAA when the culture stops growth.
The level of total IAA increases up to a maximum approx-
Figure 2. Comparison of the levels of free and total IAA when
calculated on a per gram dry weight basis (A) and a per gram fresh imately 6 to 8 d after subculture and then decreases by
weight basis (6) during the growth and maturation of Z. mays kernels approximately one-half by d 10 (Table I). Tablt! I also shows
in situ. Vertical bars indicate the SE of four replicate samples. the levels of IAA on a per gram fresh weight tlasis and on a
per gram dry weight basis. Presenting the datil on a ng per
flask basis clearly shows that Z. mays endosperm cells in
suspension cultures, without aromatic nutrient!;, are produc-
2A). When the levels of free and total IAA are plotted on a ing IAA. During the 6 d from 2 to 8 d after subculturing,
per gram fresh weight basis, it can be seen that the concen- approximately 9.5 pg of IAA were produced per flask, yield-
tration of IAA remains relatively constant after d 20 (Fig. 2B). ing a biosynthetic rate of approximately 2.5 ng g- fresh
Compared to the amount of total IAA, the free IAA levels weight h-I. This is 1.3% of the observed accumulation rate
remain relatively constant at about 1 to 2% of the total IAA. for intact kernels on the plant. Preliminary experiments in-
Considered alone, the free IAA undergoes a large increase dicate that a large amount (up to 20 pg/flasik) of IAA is
between 5 and 20 DAP from 0.018 pg kemel- (2.5 p ~ to) excreted into the media. Therefore, it is likely that the bio-
approximately 1.00 pg kemel- (22.5 PM), a 50-fold increase
on a pg kemel- basis but only a 10-fold increase on a molar
basis (Fig. 3). The amount of free IAA then remains at a
relatively constant level for the rest of the growth period. A
plot of molar IAA concentration versus time is similar in
shape to Figure 3. During the period of rapid increase in total V I I
IAA the ratio of ester to free IAA remains constant (Fig. 4)
when computed on a fresh weight, dry weight, or per kemel
basis. Free IAA constitutes approximately 1 to 2% of the
total, the remainder being ester conjugates. There was no
detectable amide-linked IAA (data not shown).
1
T 1 Table II. Synthesis of IAA and IAA esters by in vitro cultured kernels
of Z. mavs
d 20 d 32
Wgglkernel
Free 3.5 3.5
Total 16 33
Table 1. Levels of free and total IAA in Z. mays endosperm cells in suspension culture at various ages after subculturing
Days after ng flask' ng g-' fresh wt ng g-' dry wt
Subculture Free Total Free Total Free Total
2 816 f 290 2349 f 169 7 3 2 17 204 & 42 1026 & 38 2943 & 157
4 1143 -C 42 7278 & 487 64 f 8 434 f 36 1032 f 74 6726 f 421
6 865 f 249 10542 f 3318 4 0 + 15 469 f 103 754 f 219 9190 + 2881
a 890 f 163 11704 f 714 43 f 9 568 f 33 813 f 195 11028 zk 294
10 656 f 93 6018 f 421 35f6 312 f 38 *
597 123 5458 f 738
12 h 24 h Y
8 20000 -
dpm' dpm' P.
by the rate constants ko, kl, and k2. This hypothesis fits the
2.EE6
theoretical and experimental results observed in the devel- 3
li'
2.8E67 A
I
C
5
D 1
,
E 3
I
.
\
lE9
~ ~~ ~~
MasslCharge
Table IV. Comparison of the IAA biosynthetic rates calculated for
severa/ in vitro corn endosperm systems to that of the intact kernel
Tissue Rate Activity'
1 E3
ng g-' fresh wt h-' % Q I.
80 180 120 140 160 188
lntact kernel 190 1O0 Mass/Charge
In vitro kernel 99 52
Endosperm suspension 9.2 4.8 Figure 7. A, GC total ion chromatogram (TIC) of HPLC fraction
culture containing t h e methylated IAA purified from homogenized endo-
Homogenized endosperm 8.6 4.5 sperm incubated with [14C]anthranilicacid. B, Mass spectrum of
the single peak observed in A. C, Mass spectrum of methylated
a Activities are normalized to that of t h e intact kernel.
IAA.
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Copyright 1994 American Society of Plant Biologists. All rights reserved.
IAA Biosynthesis in Zea mays Kernels 349
Transport
IAA
' Catabolism
was fed, no conversion was observed at any feeding level.
On the basis of pool size and the amount of labeled IAA
produced they concluded that Trp was not a significant
Figure 8. lnputs and outputs to the pool of free IAA in plant tissues. precursor of IAA in Lemna. In most in vitro experimental
The symbols ko through k6 represent the rate constants for the systems studied thus far, less than 0.05% of the applied
indicated reactions. The concentration of free IAA is determined radioactive precursors were converted to IAA. Using the in
by the rate constants for inputs and outputs operating in the system vitro homogenized com endosperm, we observed conversion
being examined. rates of up to 8%.
Many of the early studies of IAA biosynthesis did not use
rigorous methods to positively identify the metabolite(s) of
and biochemistry (Shannon, 1982). A graph of IAA levels in interest. As a result, conclusive proof that the radioactivity
endosperm cultures is very similar to that for IAA levels in associated with that metabolite is actually due to labeled
developing kemels (Jensen and Bandurski, 1991) (Fig. 1A; metabolite has often not been presented. When one uses
Table I); therefore, the cultured cells appear to retain the radioactive precursors, the product(s) first needs to be puri-
characteristics of the intact kemel with respect to IAA dynam- fied to a constant specific activity to ensure that the radioac-
ics. In the developmental study the whole seed was assayed; tivity is associated with a specific peak. The compound must
however, it has been shown that only trace amounts of IAA then be positively identified by m a s spectral or some other
occur in the embryo and scutellum. Although IAA biosyn- qualitative analysis. By adding [3H]IAAto our sample during
thetic capacity is only 4.8% of that of the intact kemel on a extraction and then purifying to a constant 3H/14Cratio, we
per gram fresh weight basis, this is sufficient to make cultured conclude that we have indeed observed the metabolic con-
endosperm cells a suitable system for the study of IAA version of [14C]anthranilic acid to [14C]IAA. Mass spectral
metabolism. A major difference between intact kemels and analysis provided the final proof as to the identity of the 14C-
the cultured cells is the percentage of IAA in the ester form. labeled IAA.
In the developing kemels, ester IAA is maintained at approx-
imately 98% of the total for most of the developmental period The Kernel as the Site of IAA Production
(Fig. 4). In the suspension cultures, the ester IAA accounts
for approximately 65% of the total initially and then varies The seed as a site of auxin production has been proposed
between 88 and 92% of the total IAA for the remainder of for many years (Nutman, 1939; Muir, 1942) but has largely
the culture cycle. The percentage of ester after 4 d is similar been ignored as a tissue for use in IAA biosynthesis experi-
to that in vegetative tissue. This suggests that in the cell- ments. Hatcher (1943), working with rye, concluded that the
suspension cultures a11 of the rate constants ko through k6 grain was the site of auxin production based on the fact that
from Figure 8 are involved in regulating the free IAA concen- prematurely harvested grains continued to accumulate auxin.
tration, in contrast to that proposed for the kemel. This may We believe the evidence in this paper strongly supports the
be the result of IAA being utilized during cell division. In the contention that the large amount of IAA appearing in the
kemel cell division stops approximately 10 DAP. This differ- kemel is produced within the endosperm. This conclusion is
ence in ratios, however, has no impact on the usefulness of based on the following data. First, in vitro cultured kemels
this system in examining the biosynthetic pathway because were able to accumulate large amounts of IAA. Since only
of the large amount of IAA produced by the in vitro grown inorganic salts, SUC,thiamine, and Asn are present in the
cells. When studying regulation, however, there is clearly a media, the increase in the levels of IAA is direct proof that
difference in how the two systems regulate IAA levels. With de novo synthesis has occurred in situ.
the in vitro grown kemels the higher proportion of free IAA Second, the biosynthetic rate observed for the homoge-
may be a consequence of growing in culture or the different nized endosperm was 4.5% of the intact kemel and is thus a
variety of maize used. substantial fraction of the in situ rate (Table IV). This value
was determined under assay conditions of limited substrate
Corn Endosperm as a Model System for Studying IAA availability and with no correction for isotope dilution and
Biosynthesis is, thus, an underestimate of the actual biosynthetic rate.
When one evaluates the physiological importance of any Contrast between a Monocotyledonous and a
proposed IAA biosynthetic pathway, the ability of the path- Dicotyledonous Plant
way to satisfy the needs of the plant must be determined.
The pool sizes of the potential precursors and products should It has been reported by Bialek and Cohen (1989) that
be determined so that the relative importance of a compound during bean seed development the level of total IAA on a
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Copyright 1994 American Society of Plant Biologists. All rights reserved.
350 Jensen and Bandurski Plant Physiol. Vol. 106, 1994
per seed basis remains fairly constant, while the percentages Springer-Verlag, Heidelberg, Germany, pp 341-352
of free, ester, and amide IAA change. This is different from Bialek I<, Cohen JD (1989) Free and conjugated indole-3-acetic acid
m developing bean seeds. Plant Physiol91: 775-772
conclusions presented here for maize. A possible explanation Bialek I(, Michalczuk L, CohenJD (1992) Auxin biosynthesis during
for t h e lack of change i n t h e amount of total IAA i n bean seed germination in Phaseolus vulgaris. Plant Physiol 1 0 0 509-517
seeds could b e that the IAA is produced elsewhere in the Chen KH, Miller AN, Patterson GW, Cohen JD (1988) A rapid and
plant a n d then transported to t h e seed. For example, in simple procedure for purification of indole-3-aceti: acid prior to
another dicotyledonous plant, a study of the location of GC-SIM-MS analysis. Plant Physiol86 822-825
Cholodny N (1935) Uber das Keimungshormon von Gramineen.
indole alkaloid biosynthesis in lpomoea concluded that alka- Planta 2 3 289-312
loids were produced i n the leaves and then transported to Cohen JID, Baldi BG, Slovin JP (1986) '3C6-[Benzeneingl-indole-3-
t h e seed (Mockaitis e t al., 1973). We believe that i n maize t h e acetic acid. A new intemal standard for quantitativc mass spectral
indole ring is produced in t h e kemel, a n d this may be an analysis of indole-3-acetic add in plants. Plant Phyjiol 8 0 14-19
Cohen JD,Bandurski RS (1978) The bound auxins protection of
intrinsic difference between monocots and dicots. More re- indole-3-acetic acid from peroxidase-catalyzed oxidation. Planta
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provides t h e IAA for t h e developing bean seedling. In this Cohen JD, Bandurski RS (1982) Chemistry and physiology of the
system, IAA conjugates in t h e seed have little or no role in bound auxins. Annu Rev Plant Physiol33 403-43Cl
supplying IAA for t h e seedling (Bialek e t al., 1992). Again, Corcuera LJ (1967)Estudio de indoleketilinositide en g a n o de maiz.
PhD thesis, Universidad Catolico de Chile, Santiago, Chile
this is different from our findings for maize s e e h g s in Epstein E, Cohen JD, Bandurski RS (1980) Concentration and
which there is no de novo biosynthesis and the seedling is tumo'ver of indoles in germinating kemels of Zea mays L. Plant
totally reliant on IAA from conjugates stored in t h e seed Physiol65 415-421
(Jensen and Bandurski, 1990). This may explain t h e large Felker IFC, Miernyk JA, Crawford GC (1989) Characterization of
maize endosperm-derived suspension cells through out the culture
difference i n total IAA found in bean versus c o m seeds (170 cycle and enhancement of tissue friability. Plant Cell Tissue Org
ng/seed versus >160,000 ng/seed, respectively). Cult 18: 153-165
Gegenbach BG (1977) Development of maize carppsis resulhg
form in vitro pollination. Planta 134 91-93
Maize Endosperm as a System for Study of IAA Hatcher ES, Gregory FG (1941) Auxin production during the devel-
Biosynthesis opment of the grain of cereals. Nature 148: 626
Hatcher ESJ (1943) Auxin production during development of the
Because of t h e high rates of synthesis, the stability of the grain in cereal. Nature 151: 278-279
biosynthetic system to freezing, the known rates of i n situ Heyn ANJ (1935) The chemical nature of some growth hormones as
synthesis, and the availability of unlimited amounts of en- detennined by the diffusion method. Proc K Ned Akad Wet 38:
1074- 1081
zymatic material, we suggest that the endosperm of Z. m a y s Jensen lPJ, Bandurski RS (1990) Characterization by NMR of tryp-
is a suitable material for study of the mechanism(s) of IAA tophan isolated from seedlings of Zea mays grown on 30% deute-
biosynthesis. rium oxide (abstract No. 401). Plant Physiol93 5-69
Jensen PJ, Bandurski RS (1991) IAA and IAA ester content of
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ACKNOWLEDCMENTS 9 6 S-28
Jensen PJ, Momonoki Y, Bandurski RS (1992) Indole-3-acetic acid
We thank Dr. Jack Shannon for providing the endosperm suspen- biosynthesis in maize endosperm (abstract No. 374). Plant Physiol
sion cultures, Dr. Fred Below for the kemel culture material, Dr. 9 9 S-63
Yoshie Momonoki for experimental assistance, and Dr. Carolyn Kesy JM,and Bandurski RS (1990) Partia1 purification and charac-
Jensen for her assistance in preparation of this manuscript. terization of indol-3-ylacetylg1ucose:myo-inositol indol-3-ylacetyl-
transferase (indoleacetic acid-inositol synthase). Plint Physiol 9 4
1598-1604
Received February 25, 1994; accepted May 31, 1994. Kosuge T, Heskett MG, Wilson EE (1966) Miaobial synthesis and
Copyright Clearance Center: 0032-0889/94/106/0343/09. degradation of indole-3-acetic acid. I. The conversi(mof L-trypto-
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