' PATTERNS OF DEVELOPMENT AND EFFECTS OF '

BENZYLADENINE (6 - BE‘NZYLAMINQPURINE) ON THE, ‘ '

DEVELOPING ENDOSPERM 0F CORN (Zea mays L.)

Thesis for 'the Degree of M. S.
MICHIGAN STATE UNWERSITY
’ CARL RAY REED
1975

.4 ~.a

JHEEIS

 

ABSTRACT

PATTERNS OF DEVELOPMENT AND EFFECTS
OF BENZYLADENINE (6-BENZYLAMINOPURINE) ON
THE DEVELOPING ENDOSPERM OF CORN (Egg gays p.)
By

Carl Ray Reed

Several enzymes able to hydrolyse a-N-benzoyl-dl-
arginine-p-nitroanilide (BAPA) were extracted from the endo-
sperm of developing corn (Zea mays £.). They were unable to

hydrolyse bovine albumin, wheat (Triticum aestivum E.) gliadin,

 

zein, and Azocoll, and at least some were inhibited by an en-
dogenous trypsin inhibitor. Patterns of development from 13
to 41 days after pollination indicated that a) lysine and
trypsin inhibitor content did not appear to be physiologi-
cally related, b) higher trypsin inhibitor levels often found
in Opaque-2 varieties may be due to higher albumin levels,
and c) a physiological role for the trypsin inhibitor-pepti-
dase interaction in the endosperm was not apparent from this
study. The normal line was lower in peptidase, lysine, tryp—
sin inhibitor, and moisture than the genetically similar
Opaque-2 line.

Applying benzyladenine (6-benzylamin0purine) ten days

after pollination affected the rate of protein synthesis

Carl Ray Reed

relative to carbohydrate accumulation in the developing endo-
sperm of corn (Zea mays L.) grown in the field. This benzyl-
adenine treatment also affected moisture content, with the
Opaque-Z variety responding longer than its normal isoline.
Benzyladenine applied seventeen days after pollination in-
duced changes in peptidase activity. The effect of the treat—
ment was to accelerate normal developmental trends in all
cases. Data are consistent with the theory that endogenous
plant growth substances influence the sequence of events with-

in the developing endosperm of corn.

PATTERNS OF DEVELOPMENT AND EFFECTS
OF BENZYLADENINE (6-BENZYLAMINOPURINE) ON

THE DEVELOPING ENDOSPERM OF CORN (Zea mays E.)

BY

Carl Ray Reed

A THESIS

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

MASTER OF SCIENCE

Department of Crop and Soil Science

1975

ACKNOWLEDGMENTS

Sincere appreciation is expressed to Dr. Fred Elliott,
Dr. Dale Harpstead, Dr. Bernard Knezek, and Dr. Maurice
Wiese for their assistance. To Dr. Donald Penner, whose
skilled guidance and trusted friendship have contributed
time and again to my education and this research, I offer
special thanks. I also wish to mention Mr. John Brattin,
without whose assistance I might never have begun this
master's program. Finally, to Susan, who has shared so much

...thank you.

ii

LIST OF TABLES

LIST OF FIGURES

INTRODUCTION

CHAPTER 1.

List of References

CHAPTER 2.

Abstract
Introduction

Materials and Methods

TABLE OF

LITERATURE REVIEW

PEPTIDASES AND TRYPSIN
DEVELOPING ENDOSPERM
NORMAL CORN

Results

Discussion

Literature Cited

CHAPTER 3.

Abstract
Introduction

Materials and Methods

EFFECTS OF BENZYLADENINE
OF IMMATURE SEEDS OF

C 0

Results and Discussion .

Literature Cited

CHAPTER 4.

APPENDICES

SUMMARY AND CONCLUSIONS

iii

Zea

CONTENTS

INHIBITOR
OF OPAQUE*2

ON THE ENDOSPERM

mays L.

Page

iv

20
20
20
22
25
27

42

45
45
45
47
48
56
58

60

LIST OF TABLES

Page
1. Inhibition of corn peptidases by extract contain-
ing endogenous trypsin inhibitor . . . . . . . . 28
2. Correlation between parameter values . . . . . . 29

iv

LIST OF FIGURES

CHAPTER 2

1.

Peptidase extracted at pH 9.0, showing activity
per mg water~soluble protein as a function of
the pH of the assay solution . . . . . . . . .

Peptidase extracted at pH 5.0, showing activity
per mg water-soluble protein as a function of
the pH of the assay solution . . . . . . . .

Activity per gm of flour of peptidase extract-
ed at pH 9.0 . . . . . . . . . . . . . . . . .

Activity per gm of flour of peptidase extract—
ed at pH 5.0 . . . . . . . . . . . . . . . . .

Activity per mg of total protein of peptidase
extracted at pH 9.0 . . . . . . . . . . . . .

Activity per mg of total protein of peptidase
extracted at pH 5.0 . . . . . . . . . . . . .

Trypsin inhibitor activity per 0.1 mg of flour
of solutions extracted at pH 2.0 to 10.0 . . .

Trypsin inhibitor activity per mg water-
soluble protein of solutions extracted at pH
2.0 to 10.0 . . . . . . . . . . . . . . . . .

Trypsin inhibitor per gm flour over time . . .

Trypsin inhibitor per mg water-soluble protein
over time . . . . . . . . . . . . . . . . . .

Dye—binding capacity per unit of protein over
time 0 O O O O O C O O O O O O O O O O O 0

Activity per mg water-soluble protein of
peptidase extracted at pH 5.0 . . . . . . .

Percent (of the total protein present as
albumin . . . . . . . . . . . . . . . . . .

Page

33

33

33

33

35

35

35

37

37

37

37

39

14. Protein content of one gm of flour over time
15. Percent moisture over time . . . . . . . .
16. Trypsin inhibitor activity per mg total protein
over time . . . . . . . . . . . . . . . . . .
l7. Albumin content of one gm of flour over time
18. Dye-binding capacity per gm of flour as a
function of time . . . . . . . . . . . . .
CHAPTER 3
1. Total protein content of one gm of flour over
time . . . . . . . . . . . . . . . . . . .
2. The dye-binding capacity as affected by treat—
ment and time . . . . . . . . . . . . . . .
3. Trypsin inhibitor per gm of flour over time.
4. Moisture percentage as affected by treatment
and time . . . . . . . . . . . . . . . . . .
5. Peptidase activity per gm of flour over time
6. Peptidase activity per mg water-soluble

protein as affected by treatment and time.

vi

Page
39

39

39

41

41

53

53

53

53

55

55

INTRODUCTION

High crop yields have been a primary objective since
the beginning of modern agricultural research. Yet certain
aspects of yield are still poorly understood. One such as-
pect is the biochemical and physiological dimensions of the
mechanism by which certain genetic information produces high"
yielding plants. Cereal plant breeders have for decades
worked with phenotypically similar lines which varied widely
in the total weight of seed produced. Obviously, one criti-
cal difference between such genotypes involves the ability
to direct nutrients to the developing seeds.

Protein content of the seed (the amount of protein
present) is usually considered a secondary, though important
objective of most breeding programs. Protein quality, the
ability of the protein to supply essential amino acids in
proper proportions, and the ease with which the protein is
digested in mammalian systems, is a somewhat more recent con-
cern. Even today, the prospect of identifying a genotype
with protein ten percent more available than an equally pro-
ductive counterpart appears to engender less enthusiasm than
the idea of producing a ten-percent higher yielding line,
though the two are functional equivalents. The importance

of protein quality is so little understood that the market

currently provides little economic incentive for researchers
to develop, or growers to produce higher—quality protein
grain.

This study examines certain questions related to both
yield and protein quality. The first inquiry involves a
group of proteins, found in the seeds of corn (Zea_m§y§ L.)
which inhibit mammalian hydrolytic enzymes and thereby de»
crease the ability to utilize the grain nutrients. The second
involves the role of plant growth substances in the movement
of assimilates to developing seeds. Thus the focus of this
study has come to rest on the intersection of several areas
of plant science, including nutritional quality of grain,
plant andsfifixiphysiology, biochemistry, endocrinology, and
genetics. Hopefully, basic studies like this one will even-
tually lead to an understanding of the components of nutri-
tional quality, as well as to the development of new screen-
ing techniques for improved protein availability. In the
protein-starved world of 1975, such information is vital,
and must quickly find use in plant breeding programs and
food processing technologies.

Specifically, this study investigates the function of
trypsin inhibitors in the endosperm of two isogenic lines
(normal and Opaque~2) of commercial hybrids. Benzyladenine,
a compound structurally similar to zeatin (a cytokinin from
corn) was applied to immature ears of both lines to determine
whether the trypsin inhibitors function by protecting stor-

age proteins from endogenous proteases during seed filling

and dormancy. Benzyladenine promotes the accumulation of nut-
rients through the symplast, and may be involved in synchro-
nizing translation of genetic information. Since the levels
and normal developmental patterns of trypsin inhibitor and
other parameters in the endosperm of these seeds were known,
it was hoped that benzyladenine-induced changes in protein
fractions could elucidate the function of the trypsin in»

hibitor.

CHAPTER 1

LITERATURE REVIEW

Many of the topics approached in this research, includ-
ing seed proteases and trypsin inhibitors, nutritional quali-
ty and protein availability, cytokinins, and the genetics of
high-lysine maize have been active areas of research for many
years. These inquiries have been productive, and the litera-
ture generated by them is voluminous. This review will at-
tempt to touch on significant accounts from these areas, but
will be generally confined to research on crop plants.

Seeds, and many organs of mature crop plants contain
toxic substances. Recently summarized by Liener (52), these
include protein and non-protein enzyme inhibitors, hemagglu-
tinins, thiolglucoside goitrogens, cyanogens, carcinogenic
cycasin, saponins, and phytates. In the basic cereal grains,
the most intensively studied of these toxins are the proteo-
lytic inhibitors, recently reviewed by Ryan (90).

The first seed protease inhibitor was discovered in
wheat in 1938 (86), and first isolated in pure crystal form
(from soybeans) by Kunitz (50) just 30 years ago. Since then
protease inhibitors have been found in the seeds of so many
genera that they appear to be almost universal (2, 6, 19, 21,
28, 37, 40, 48, 49, 57, 61, 89, 95, 116, 125).

4

Two theories of the function of protease inhibitors in
seeds have been proposed. One states that protease inhibi-
tors protect seed nutrient reserves against attack by insect
and microbial predators (90, 116). This theory has been ex-
plored by several investigators, who have found that proteo-
lytic inhibitors from seeds do inhibit the digestive enzymes
of certain insects (4, 5, 8, 9). The question remains, how-
ever, whether this "protective" function of protease inhibi-
tors can fully account for the presence of the inhibitors in
such a wide variety of plants.

An intriguing phenomenon explored by Green and Ryan
(28) and McFarland and Ryan (56) is the production of pro—
tease inhibitor in wounded leaves. Since wound-induced pro—
tease inhibitor is synthesized in intact cells somewhat re-
moved from the injury, some unknown factor must move from
wounded cells to induce protease inhibitor synthesis. Thus
the wound response—-of obvious value in case of insect
attack-~appears to support the "protective" theory of pro-
tease inhibitor function.

The second theory is that protease inhibitors may be
involved in the regulation of endogenous proteases (90).
Though most of the known protease inhibitors have not been
shown to inhibit endogenous proteases (90), some such inter-
actions have been documented (12, 21, 95). In germinating
lettuce seeds the disappearance of protease inhibitor cor-

responds with the activation of proteolytic activity in a

manner suggesting that the protease inhibitor may function in
the mechanism of germination (95, 96). Little evidence exists
to indicate such a relationship in other species.

The review by Ryan (90) also summarized research on
seed proteases,reports of which are found in eighteenth cen-
tury literature (90). Though proteases are known to exist in
certain plant organs (75, 99), all seeds are assumed to con—
tain proteases during germination (3, 7). The origin of
these enzymes is unknown in many species (10, 24, 43, 83, 84).
In other species they are present in active form in dry seeds
(11, 12, 27, 58, 71, 81, 82, 91, 95, 101). Some are acti-
vated during germination (1, 26, 94, 104, 105, 106, 127), and
others are synthesized g3 2939 after inbibition (34, 35, 41,
109, 128). Some are able to hydrolyse intact storage protein
(26, 77, 83, 84, 105), while others appear to be specific for
smaller peptides (11, 24, 27, 58, 80, 81, 91).

Cytokinins delay leaf and plant senescence, apparently
by slowing the rate of net protein degradation (14, 38, 50,
65, 67, 78, 88, 100, 103, 108, 110, 111, 113, 114, 117), and
induce nutrient mobilization to the treated areas of leaf
(55, 62, 68, 69, 76, 119) and stolon (102) tissue. Young ex-
panding grape leaves sprayed with cytokinin solution accumu-
late photosynthate faster than untreated leaves, but mature
leaves respond only when shaded (85). Cytokinin increases
the rate of cell division and expansion in callus tissue (19,
31, 32), and interacts with other plant growth substances to

control growth and differentiation of callus and cut stems

(31, 107, 121, 123). In some germinating seeds, rates of
hydrolysis are affected by cytokinins and other plant growth
substances (41, 77, 97, 115, 128), but in immature seeds,
where these hormones are known to exist at relatively high
levels (13, 63, 64, 65, 92, 112), and where assimilates are
concentrated in a manner similar to the cytokinin~induced
phenomenon (16, 54, 118, 120, 121), little is known of their
function. It is, however, generally assumed that cytokinins
interact with other plant growth substances to regulate meta-
bolic activity and development in seeds (7, 22, 79).

Corn is an inferior source of protein for monogastric
animals (29, 36, 73, 74, 98). The endosperm of commonly-
grown varieties contains accumulations of a well-characterized
trypsin inhibitor (39, 40), but the nutritional significance
of this heat-stable protein is unknown. Trypsin inhibitor
from soybean has been shown to account for 40% of the growth
depression observed in rats fed unheated soybean meal (25,
45, 46, 47, 53, 86, 93). Obviously, the possibility that the
trypsin inhibitor contributes to the poor quality of corn
protein cannot be discounted.

Another facet of the problem of corn protein availa-
bility to monogastric systems is that much of the endosperm
protein of normal corn is present in the prolamine fraction
(17, 20, 33, 42, 72). Corn prolamine (zein), besides being
severely deficient in lysine and tryptophan, is resistant to
protease attack due to its tertiary structure and the pres-

ence of large numbers of disulfide bonds (23, 44). The

Opaque-2 gene has received much attention and stimulated much
research because it modifies the protein fractions of the en-
dosperm to produce a higher lysine, lower zein seed (15, 17,
42, 59, 60, 70, 124, 125). However, certain Opaque-2 va-
rieties, perhaps because of higher trypsin inhibitor levels
(32), have performed poorly in feeding trials (Elliott--

personal communication).

10.

11.

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19

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CHAPTER 2

PEPTIDASES AND TRYPSIN INHIBITOR IN THE
DEVELOPING ENDOSPERM OF OPAQUE-Z AND NORMAL CORN

Abstract

Several enzymes able to hydrolyse d-N-benzoyl-dl-
arginine-p—nitroanilide (BAPA) were extracted from the endo-
sperm of developing field-grown corn (Lea mays L.). They
were unable to hydrolyse bovine albumin, wheat (Triticum
aestivum L.) gliadin, zein, and Azocoll, and at least some
were inhibited by the endogenous trypsin inhibitor. Patterns
of development from 13 to 41 days after pollination indicated
that a) lysine and trypsin inhibitor content did not appear
to be physiologically related, b) higher trypsin inhibitor
levels often found in varieties with the Opaque-2 gene may be
due to higher albumin levels, and c) a physiological role for
the trypsin inhibitor-peptidase interaction in the endosperm
was not apparent from this study. The normal line was lower
in peptidase, lysine, trypsin inhibitor, and moisture than

the genetically similar Opaque-2 line.

Introduction

 

In attempts to upgrade the quality of plant protein for

consumption by humans and other monogastric animals, scientists

20

21

have explored several aspects of plant protein utilization.
The essential amino acid profile as determined from hydroysates
and the presence of antimetabolites are two such aspects.

In corn (Lea mays L.) the Opaque-2 gene influences
the proportions of certain protein fractions and increases
the quantity of lysine and tryptophan present in the protein
(4, 15). Certain Opaque-2 varieties may possess undesirable
characteristics as well, including higher levels of trypsin
inhibitor activity (10). Trypsin inhibitor is present in re-
latively high levels in the mature endosperm of normal corn,
and may limit protein efficiency and be partially responsible
for the relatively low availability of lysine (9, 10, 16).
With soybean [Glysine max. (L.) Merr.] trypsin inhibition
accounts for forty percent of the growth depression observed
in rats (17, 18).

The question arises whether a high level of trypsin in-
hibitor is of physiological importance in a high lysine, low
zein seed, or merely a side effect of the altered proportions
of the protein fractions of the Opaque-2 seed. This poses
the larger question of the significance of proteolytic in-
hibitors in seed physiology.

Seed proteases include those present in dry seeds (25,
27), those activated during germination (30), and those
synthesised de Egyg during germination (12, 14). Proteases
are also found in non-seed organs of plants (31). Some are
able to hydrolyse intact storage protein (22, 23, 27, 28),

while others appear to be specific for smaller peptides (3).

22

Some are inhibited by endogenous or exogenous protease in-
hibitors (6, 29). Others are not 0, 14, 28).

This study examined the relationship between proteolysis,
trypsin inhibitor, and lysine content in the developing endo-

sperm of genetically similar Opaque-2 and normal corn.

Materials and Methods

 

Isogenic lines of Funk's special cross hybrid corn
G4384-A (normal endosperm) and #26536 (Opaque endosperm) were
grown during 1974 at the Michigan State University Crop Science
research plots at East Lansing. Rows 110 M long spaced 102
cm apart were planted at 9,100 plants/ha to provide approxi-
mately 100 plants of each line per plot. A split plot design
was used, with four replications. Forty plants were chosen
at random from each plot, with twenty from each line. First
ears appearing on the stalk were tagged when the silk was
1.3 cm long on August 18 to establish the pollination date.
Open pollination was allowed. Five ears were harvested from
each sample at 13, 20, 27, and 41 days after pollination.
Husk and leaf tissue was removed from the ear at harvest and
the distal half of the ear immediately placed on ice.

Endosperm samples were obtained with a corn cutter ad-
justed to remove the tops of the kernels only. Seeds from
about 2.5 cm from the tip to mid-ear were included in the
sample. Material from each of the five ears from each plot

was bulked and freeze-dried. The dry sample was ground,

23

sealed,and stored in a desiccator for analysis.

Water-soluble protein was determined by a modification
of the method of Lowry, Rosebrough, Farr, and Randall (19).
Five concentrations of extract from each sample were analyzed.
The protein content was calculated from the regression line
of optical density plotted against concentration and taken as
the sample value. Total protein was determined by Kjeldahl
digestion followed by a modified Nesslerization using Sigma
Ammonia Color Reagent from Sigma, Co. The value 6.25 was
used to convert mg nitrogen to mg protein. Percent moisture
was determined by pre- and post-freeze-dry weight.

To extract the peptidase, endosperm flour was weighed
into centrifuge tubes, distilled water was added and the pH
was adjusted with l M NaOH or 1M HCl after thorough mixing.
After two hours of extraction at 4 C without further agita-
tion, the preparation was centrifuged for 15 min. at 17,300
x G (O C). The supernatant fluid was decanted, diluted one
to one with distilled water, and used as the crude enzyme
preparation.

Peptidase activity was measured using a modification of
the method of Erlinger, Kokowsky, and Cohen (5) and Burger
and Siegelman (1), employing BAPA (a-N-benzoyl-dl-arginine-
p-nitroanilide HCl) from Sigma, Co. as substrate. Two test
tubes, each containing 1 ml distilled water and 1 ml enzyme
preparation, and one tube containing the water, enzyme, and
1 ml of 30% acetic acid were prepared for each sample. The

tube containing acid provided a blank for each sample, while

24

the others gave a replication of each value. After equili-
bration for 2 min. at 37 C in a water bath, the assay was
started by adding 7 ml of 0.05 M tris buffer (0.01 M CaClZ)
of appropriate pH value, and containing 3 x 10—4gm BAPA/ml.
After 10 min. at 37 C, the reaction was stopped with the ad-
dition of 1 ml of 30% acetic acid. The solutions were allow-
ed to stand for at least 20 min. at room temperature, then
the optical density was determined at 4101m1and compared
against the appropriate blank. The average of the two values
was compared to a stock trypsin solution run with each sample
and peptidase activity was calculated. In this paper, the
change in optical density due to the peptidase activity is
referred to as A O. D.

Trypsin inhibitor was extracted similarly to peptidase
at pH 5.0, but after centrifugation, the crude supernatant
fluid was held at 80 C for 10 min. to destroy peptidase ac-
tivity. The resulting suspension was centrifuged at 12,100
x G for 10 min., decanted, and the supernatant fluid diluted
to a concentration affording 50% inhibition. One ml of
stock trypsin solution, prepared by dissolving 5 mg trypsin
(2X crystallized, Worthington Biochem. Corp.) in 100 m1 0.001
M HCl, was added to each of two tubes containing the inhibitor
preparation, and one tube containing the inhibitor and 1 ml
of 30% acetic acid. The assay described above for peptidase
activity was followed. Optical density was determined, per-
cent inhibition was calculated from a point near 50%, and

expressed as ug trypsin inhibited per 10 ml of the described

25

solution under assay conditions.

Lysine estimates were made using a modification of the
Udy dye-binding technique (32). Samples were weighed to pro-
vide 65 mg protein per sample. Forty m1 of dye solution con-
taining 1.3 gm Acid Orange-12 dye/L were added and the mix-
ture was agitated for 1 hour. Solutions were filtered and
percent transmission determined on a Udy color analyzer.
Values, called the Dye-Binding Capacity (DBC) are expressed
as gm dye bound/L. Individual sample values reported here
are means of two observations.

Values reported in Figures 3 through 6 and 9 through
18 are means of four individual sample values for days 20
and 41, and eight sample values for days 27 and 34. Single
sample values are reported for day 13. Figures 1, 2, 7 and
8 show individual values, though all such experiments were re-

peated at least once with similar results.

Results

Extractions from normal corn at pH 5.0 and 9.0 were
assayed with BAPA for peptidase activity. The results in-
dicated the presence of at least five activity optima, es-
timated at pH 4.0, 5.5, 7.5, 8.4, and 10.0 (Fig. l). The
broad peak apparent when the endosperm was extracted at pH
5.0 (Fig. 2) may be interpreted as possibly consisting of
the three alkaline peaks extracted at pH 9.0. Presumably,

the peak at pH 7.5 was the same enzyme reported by Melville

26

and Scandalios (21), extracted in that laboratory at pH 7.5.

These peptidases were unable to hydrolyse zein, wheat

(Triticum aestivum L.) gliadin, bovine albumin, or the gen-

 

eral proteolytic substrate Azocoll (Calbiochem.). In sub-
strate specificity, therefore, they resemble peptidases ex-
tracted from the roots of soybean seedlings (7), the embryos

of germinating barley (Hordeum vulgare L.) and wheat (2, 3,

 

24), and seeds of Scots pine (pinus sylvestris) (26).

 

The peptidase activity extracted at pH 5.0 and the pH
9.0-extracted enzyme whose activity peak is at pH 8.4 were
selected for the development study. One gm and 0.25 gm, re-
spectively, of endosperm flour in 10 ml distilled water were
used to extract these enzymes. The difference in the magni-
tude of activity (Figs. 3, 4), plus the distinctively re-
versed positions of the lines at day 20 (Figs. 5, 6) show
that indeed two different enzymes are being assayed.

Abundant trypsin inhibitor was extracted over a wide
pH range from the endosperm of maturing corn, as shown in
Figure 7. Figure 8 indicates that the amount trypsin inhibi-
tor was not simply a function of the amount of albumin solu-
ble at a given pH. Trypsin inhibition was linear over a
wide range (from 30 to 65% inhibition).

Trypsin inhibitor appeared in the endosperm about 20
days after pollination and greatly increased for the next
two weeks (Fig. 9). The relative quantity of inhibitor then
declined somewhat in the normal line while continuing to

increase in the Opaque-2 line. The trypsin inhibitor fraction

27

of the soluble protein showed no such decline, but continued
to increase throughout the 21-day period (Fig. 10).

Tests of auto-inhibition were conducted using the heat-
treated extract to provide a peptidase-free trypsin inhibitor
solution, and extract from the l3-day old seeds, which con-
tained high peptidase activity but almost no inhibitor. Table
1 shows data from one of four experiments, all of which in-
dicated definite inhibition of endogenous peptidases by the
trypsin inhibitor. Inhibition was observed for all pH optima,
but the impure nature of the extract precluded the conclusion
that all peptidases were inhibited.

An unexpected result, shown in Fig. 11, is significantly
more lysine as measured by the DEC per mg protein in the nor-
mal line than in the Opaque—2 variety early in the seed fill-

ing period.

Discussion

 

Data in Table 2 show correlations between important
parameters of endosperm development examined in this study.
Forty-one day old Opaque-2 endosperm was higher than the
normal line in trypsin inhibitor, peptidase, and basic amino
acids. Since DBC and trypsin inhibitor were not correlated
within lines, and gross differences appeared in the patterns
of development of these two parameters, the relationship be-
tween basic amino acids, such as lysine, and trypsin inhibi-

tor appears to be secondary, not causal. Data in Figure 10

28

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wuflommmo mcfipcwnnwwp paw o.m mm mmmpfiummm
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muflommmo mcfipcflnnmmp paw Houwnflacfl cwmmmuu
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muflommmo mcflpcflnsmmp cam Houwnfl£CH :mmmmuu

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.cuoo mafiQon>mp aw mnmumEmumm
ucmuuomEfl cmm3umn mcoflumdonuoo coflmmwummu ummcfiq sum manna

30

show that, to day 41, trypsin inhibition per mg albumin was
the same in both lines, 1. e., the higher levels of trypsin
inhibition in the Opaque-2 must be due to higher levels of
water-soluble protein. Peptidases, however (Fig. 12), con-
stituted a larger fraction of the albumin in the Opaque-2
line than in the normal line. ,

Jimenez (15) found the albumin fraction of mature seeds
.to be four times greater in the opaque 2 than in the normal
line, a trend not observed in this study. However, large
variations in albumin levels from replication to replication
were observed in this study, especially in the very young
material. These variations may have masked differences be-
tween lines. Definite differences between lines can be ob-
served in the development of protein fractions over time
(Fig. 13) in agreement with the fractionation study of Dalby
(4).

Judging from the specificities of the peptidases, their
function probably involves normal protein turnover rather
than hydrolysis of storage products. A physiological function
of the trypsin inhibitor in the endosperm is difficult to en—
vision. Since reaction with the peptidase destroys inhibi-
tory activity (13), some physical separation of the trypsin
inhibitor from the peptidase must exist in order for accumula-
tion of the trypsin inhibitor to occur. Comparing the data
of Melville et a1. (21) with that of Harvey and Oaks (11) one
finds that trypsin inhibitory activity is lost from corn endo-

sperm during germination at a rate somewhat slower than that

31

for total nitrogen. This indicates that the inhibition of
endogenous peptidases by the trypsin inhibitor located in the
endosperm is probably not a central part of the mechanism of
germination in corn. Trypsin inhibitors may serve simply as
storage proteins. An alternate theory is that some molecules
of the inhibitor are arranged in such a way as to protect
against either premature release of some lysozyme-like compo-
nents activated during germination, or the degradation of the
lysozyme-synthesizing apparatus. Finally, one may postulate
that, while serving as a storage protein in the endosperm,
the trypsin inhibitor may function a) in or near the embryo,
or b) in some essential mechanism, such as the wound response
of leaves (8, 20), in one or more of the plant organs from

which it has been extracted.

 

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:Hmum3 mE umm >uw>fluom mcfl3o:m

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32

s: 5'3

figvd

Peptidase activity
A, O. 9.
85353883

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34

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Trypsin inhibition
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36

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38

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percent
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Igrcent

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40

Figure 17--Albumin content of Figure 18-—Dye-binding capacity
one gm of flour per gm of flour over

over time. time.

.1

 

34 41
days after pollination

27

20

 

 

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27

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10.

Literature Cited

 

Burger, W. C., and H. W. Seigelman. 1966. Location of
protease and its inhibitor in the barley kernel.
Physiol. Plant. 19:1089-1093.

Burger, W. C., N. Prentice, J. Kastenschmidt, and J. D.
Huddle. 1966. Partial purification of proteases
from germinating barley. Cereal Chem. 43:546-554.

Burger, W. C., N. Prentice, J. Kastenschmidt, and M.
Moeller. 1968. Partial purification and charac-
terization of barley peptide hydrolases. Phytochem.
7:1261-1270.

Dalby, A. 1966. Protein synthesis in maize endosperm.
In: E. T. Mertz and O. E. Nelson, eds., Proc. High-
lysine Corn Conf. Corn Corn Ind. Res. Found., Inc.,
Washington, D. C. pp. 80-89.

Erlanger, B. F., N. Kokowsky, and W. Cohen. 1961. The
preparation and properties of two new chromogenic
substrates of trypsin. Arch. Biochem. Biophys. 95:
271-278.

Filho, J. X. 1973. Trypsin inhibitors during germina-
tion of Vigna sinensis seeds. Physiol. Plant. 28:
123—132.

 

Graf, G., and R. E. Hoagland. 1969. Partial purifica-
tion and characterization of an amidohydrolase from
soybean. Phytochem. 8:827-830.

Green, T. R., and C. A. Ryan. 1973. Wound-induced
proteinase inhibitor in tomato leaves. Plant Physiol.
51:19-21.

Gupta, J. D., A. M. Dakroury, A. E. Harper, and C. A.
Elvehjem. 1958. Biological availability of lysine.
J. Nutr. 64:259-270.

Halim, A. H., C. E. Wassom, and H. L. Mitchell. 1973.

Trypsin inhibitor in corn (Zea mays L.) as influenced
by genotype and moisture stress. Crop Sci. 13:405-407.

42

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

43

Harvey, B. M. R., and A. Oaks. 1974. The hydrolysis
of endosperm protein in Zea may . Plant Physiol.
53:453-457.

Harvey, B. M. R., and A. Oaks. 1974. Characteristics
of an acid protease from maize endosperm. Plant
Physiol. 53:449-452.

Hochstrasser, V. K., M. Muss, and E. Werle. 1967.
Reindarstellung und charakterisierung des trypsin-
inhibitors aus mais. Hoppe-Seyler's Z. Physiol.
Chem. 348:1337-1340.

Jacobsen, J. V., and J. E. Varner. 1967. Gibberellic
acid-induced synthesis of protease by isolated
aleurone layers of barley. Plant Physiol. 42:1596-1600.

Jimenez, J. R. 1966. Protein fractionation studies of
high lysine corn. In: E. T. Mertz and O. E. Nelson,
eds., Proc. High-lysine Corn Conf., Corn Ind. Res.
Found., Inc., Washington, D. C. pp 74-79.

Kakade, M. L. 1974. Biochemical basis for the differ-
ences in plant protein utilization. J. Agric. Food
Chem. 22:550-555.

Kakade, M. L., D. E. Hoffer, and I. E. Liener. 1973.
Contribution of trypsin inhibitors to the deleterious
effects of unheated soybeans fed to rats. J. Nutr.
103:1772-1778.

Kakade, M. L., N. Simons, and I. E. Liener. 1970.
Nutritional effects induced in rats by feeding natural
and synthetic trypsin inhibitors. J. Nutr. 100:
1003-1008.

Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J.
Randall. 1951. Protein measurement with the folin
phenol reagent. J. Biol. Chem. 193:265-275.

McFarland, K., and C. A. Ryan. 1974. Proteinase in-
hibitor-inducing factor in plant leaves. Plant
Physiol. 54:706-708.

Melville, J. C., and J. G. Scandalios. 1972. Maize
endopeptidase: genetic control, chemical characteri-
zation, and relationship to an endogenous trypsin in-
hibitor. Biochem. Genet. 7:15-31.

Parish, R. W. 1975. The lysosome-concept in plants II
location of acid hydrolases in maize root tips.
Planta 123:15-31.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

44

Penner, D., and F. M. Ashton. 1967. Hormonal control
of proteinase activity in squash cotyledons. Plant
Physiol. 42:791-796.

Prentice, N., W. C. Burger, J. Kastenschmidt, and J.
Huddle. 1967. The distribution of acidic and neutral
peptidases in barley and wheat kernels. Physiol.
Plant. 20:361-367.

Ryan, C. A. 1973. Proteolytic enzymes and their in-
hibitors in plants. Ann. Rev. Plant Physiol. 24:
173-196.

Salmia, M. A., and J. J. Mikola. 1975. Activities of
two peptidases in resting and germinating seeds of
Scots pine, Pinus sylvestris. Physiol. Plant. 33:
261-265.

 

St. Angelo, A. J., R. L. Dry, and H. J. Hansen. 1969.
Localization of acid proteinase in hempseed. Phyto-
chem. 8:1133-1138.

Semadeni, E. G. 1967. Enzymatische charakteresierung
der lysosomenaquivalent (spharosomen) von maiskeim-
1ingen. Planta 72:91-118.

Shain, Y., and A. M. Mayer. 1968. Proteolytic enzymes
and endogenous trypsin inhibitor in germinating
lettuce seeds. Physiol. Plant. 18:853-859.

Shain, Y., and A. M. Mayer. 1968. Activation of en-
zymes during germination--trypsin-like enzyme in
lettuce. Phytochem. 7:1491-1498.

Singh, N. 1962. Proteolytic activity of leaf extracts.
J. Sci. Food Agric. 13 325-332.

Udy, D. C. 1971. Improved dye method for estimating
protein. J. Amer. Oil Chem. Soc. 48:29A-33A.

CHAPTER 3

EFFECTS OF BENZYLADENINE ON THE ENDOSPERM
OF IMMATURE SEEDS OF Zea mays L.

Abstract

Applying benzyladenine (6-benzylaminopurine) ten days
after pollination affected the rate of protein synthesis re-
lative to carbohydrate accumulation in the developing endo-
sperm of isogenic varieties of Opaque-2 and normal corn (Lea
mays L.) grown in the field. This benzyladenine treatment
also affected moisture content, with the Opaque-2 variety re-
sponding longer than its normal isoline. Benzyladenine ap-
plied seventeen days after pollination induced changes in
peptidase activity. In all cases, the effect of the treat-
ment was to accelerate normal developmental trends. Data are
consistent with the theory that endogenous plant growth sub-
stances influence the sequence of events within the develop-

ing endosperm of corn.

Introduction

 

Integrated development of differentiating tissues,
such as young fruits or seeds, requires controlled sequential
translation of genetic information (1, 8). Since developing

seeds contain high concentrations of these plant growth

45

46

substances (5, 12, 14), it has become a common theory that
auxins, gibberillins, and cytokinins may function in the con-
trol of developmental activities (4, 8). The role of ethylene
in maturing fruit, for example, is well documented (9).

A second phenomenon associated with developing seeds
is the apparent ability to attract assimilates against a con-
centration gradient (1, 2). Since this nutrient-mobilizing
capacity can be induced in areas of detached leaves (6, 13),
intact stolons (11), and whole plants (10, 15) by applica-
tion of cytokinin, it is possible that cytokinins function
in the nutrient-attracting mechanism associated with metabolic
sinks such as young seeds. Few insights into the function and
capabilities of cytokinins relative to natural metabolic sinks
have appeared in the literature. An interesting exception is
the report by Quinlan and Weaver (10) that added benzyladenine
(6-benzylaminopurine) (BA) increased the movement of carbo-
hydrates through the symplast to young, expanding leaves,
but shade was required in addition to the BA treatment to re-
produce the effect in old, expanded leaves. The possibility
of a cytokinin-light interaction in natural sinks is es-
pecially interesting since developing seeds of most--if not
all--plants are shaded.

The objective of this research was to examine the ef-
fects of BA on developmental patterns in the endosperm of

normal and high-lysine Opaque-2 isogenic lines of corn (Lea

mays L.).

47

Materials and Methods

 

Genetically similar Opaque-2 and normal endosperm Funk's
special-cross hybrid corn varieties were provided by Dr. E.
Rossman of the Department of Crop and Soil Sciences, Michigan
State University. Rows 110 M long, spaced 102 cm apart were
planted at 9,100 plants/ha to provide approximately 100 plants
of each line per plot. A split plot design was used, with
four replications. Two treatment times, with controls for
each, were provided within each replication.

Treatments consisted of an injection of 5 ml of 1 x 10"4
benzyladine (6-benzylaminopurine) from Sigma, Co. The BA so—
lution was prepared by dissolving 11.25 mg BA in 25 ml abso-
lute ethanol, then diluting to 500 ml with distilled water.

A cylinder of tissue, through the leaves, fruits, and about
2.5 cm of cob, was removed with a size 22G hollow-point needle.
Five ml of the BA was immediately injected into the puncture.
The wound was sealed with petroleum jelly. Samples to be
treated received one such injection, at either 10 or 17 days
post-pollination. Controls received 5 m1 of a 5% ethanol so-
lution. Seeds adjacent to the wound appeared to be infected
in nearly all cases, and were excluded from the sample.

Further details of the harvest, handling of plant ma-
terial, and laboratory techniques are described in Chapter 1.

Values reported for day 13 are individual observations. All

other values are means of four sample values.

48

Results and Discussion

 

The graphs shown in Figure 1 indicate that accumulation
of the non-protein fraction was more rapid than protein syn-
thesis from 20 to 34 days post-pollination. This trend was
observed in all samples, but was significantly (5% level)
more rapid in the treated than the control seeds at day 27.
Lysine content, as measured by the dye-binding capacity (DBC)
results (Fig. 2) verify this treatment-induced phenomenon,
with treated seeds showing a significantly (5% level) lower
DBC at day 27 than the controls. Since no significant dif-
ferences between treated and control seeds in the DBC/mg pro-
tein were found, it follows that the endosperm of treated
seeds had less DBC because of less protein per gm flour, at
day 27, than untreated seeds. The first BA treatment, there-
fore,caused the trend in relative rates of protein vs. non-
protein accumulation to accelerate, such that treated endo-
sperm exhibited that protein content observed in the con-
trols only after several more days of growth. The protein
content of the untreated seeds reached its lowest value at
day 34, then began a relative gain to day 41. This shift was
anticipated by the treated seeds, especially in the normal
line, which showed a higher percent protein at 34 days post-
pollination than the controls.

Although BA-induced effects were not statistically sig-

nificant at day 23, they may help explain Figure 3, where

49

trypsin inhibitor activity was analyzed as a function of total
protein. The data indicated that variations between replica-
tions tended to be smaller for trypsin inhibitor than for most
of the other parameters, including total protein. Since no
differences in trypsin inhibitor activity were observed be-
tween treated and untreated seeds on the basis of gm soluble
protein or gm of flour, it is probably that trypsin inhibitor
activity did not differ from treated to untreated samples,
but did provide a stable reference point against which dif—
ferences in percent protein could be observed. The treated
normal line, having more protein, and about the same amount
of trypsin inhibitor at day 34, showed lower trypsin inhibitor/
mg protein than the control (Fig. 3). The Opaque-2 line is
higher in trypsin inhibitor than the normal line (Chapter 1).

Data in Figure 3 show not only that the BA caused normal
developmental patterns to accelerate, but that where varietal
differences existed, the treated seeds anticipated the pat-
tern typical of the variety. At day 34, the trypsin inhibitor
fraction of the total protein reversed its l4-day trend in the
normal control line, but continued to increase in the Opaque-2
control line. Treated endosperm exhibited exactly the same
pattern, but the rate of development again appeared to be
greater in the treated lines than in the controls.

The first injection (10 days post-pollination) also in-
fluenced the moisture content at day 27 (Figure 4). While

both lines responded at 27 days post-pollination (significant

50

at the 5% level), only the treated Opaque-2 line was differ-
ent (5% level) at day 34. Although the addition of BA caused
the Opaque-2 line to behave as normal endosperm at day 34,

it cannot be concluded that native cytokinin must control
this, or any other parameter influenced by BA. Cytokinins
interact with other hormones in both synergistic and antago-
nistic ways (4, 8), suggesting that BA treatment may have
produced the observed effects by upsetting a previously-estab-
lished hormone balance. Further, cytokinins are sometimes
altered in transport or at the site of their action (3, 6),
so the active molecule may not have been BA, but some metabo-
lite thereof.

Both parameters (protein and moisture) discussed above
first responded to the treatment 17 days after the BA was ap-
plied. Neither parameter was influenced by the second in-
jection time. In contrast, the peptidase activity (Figure 5)
was unaffected by the first treatment time, but showed a sig-
nificant (1% level) response 17 days after the second treat-
ment. The BA was deposited in the pithy rachis, and was as-
sumed to gradually migrate to areas of high metabolic activity
in the adjacent seeds. It seems likely that the 17 day lag
period observed in all affected parameters between the time
of application and the first reSponse was due to the time in-
volved in transport. Therefore, it should not be concluded
that the developmental patterns were insensitive to the BA

before this time.

 

lilill '1' I. Illa. Ill

51

Peptidase activity per gm flour (Figure 5) showed sig-
nificant results at day 34 only, but peptidase activity per
mg albumin (Figure 6) exhibited significant (5% level) BA-
induced effects for days 34 and 41. As with the percent
protein (and in contrast to percent moisture), the normal line
appeared to respond longer than the Opaque-2 variety.

It is obvious from this data that the endosperm of seeds
examined in this study contained components demonstrating de-
velopmental patterns quite different from one another. For
example, carbohydrate and protein fractions were synthesized
at different rates depending on the maturity of the seed, the
moisture percentage declined to day 20, then increased for a
time before dropping off rapidly, the peptidase fraction of
the albumin first increased, then declined, while the trypsin
inhibitor did not appear until the seed was about two weeks
old.

The results of this study suggest that the chronology
of development within the endosperm is influenced by plant
growth substances. None of the parameters examined here re-
sponded to both treatment times, indicating that different
fractions may have had different "critical periods" for the
translation of the appropriate genetic information. The ef-
fects of adding BA were consistent with the theory that the
rate of synthesis of some protein and/or carbohydrate fractions
is sensitive to BA during the period 27 to 34 days post-
pollination. In both varieties, parameters within the treated

seeds exhibited accelerated developmental trends.

.ummu omcmu OHQHDHSE m.smocso
>Q ucmnommap >HDCMUAMHGmHm no: out
.oEHu paw ucoaumoup mp mumuuoH GOEEOO nuflz mammz .Hson

monommmm mm mmmucooumm mucumHOZIIq onsmflm mo Scum mom HODHQASCH Gammmuhnum ousmflm

.HmEuoc Umpmmuul
HOEHOC poummuucso N mammmo

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.mEHD pcm DcmEpmouu an oouommmm .mEfiu Hm>o Macaw mo Emum

mm mpflommmo mcflpcflnam>p onelnm muomflm oco mo Dampcoo samuonm Hmuoella madman

52

Trypeln lnhlbi "on

pg Inhibited/In. protein

uoneumod .Ieue eAep £615

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no! "tuned .Ieue elep

it

 

 

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N O O O O
N
u 01 ‘
3,
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96
0 O a O
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um I

 

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Figure 5--Peptidase activity Figure 6--Peptidase activity
per gram of flour. per mg water-

soluble protein as

affected by treat-

ment and time.

54

55

34 41
days after pollination

l

27

 

 

,20b

re .p
1.... mm.

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11.

Literature Cited

 

Bidwell, R. G. S. 1974. Plant Physiology. Macmillan
Publishing Company, New York. pp. 634.

Mason, T. C., and E. Phillips. 1937. The migration of
solutes. Bot. Rev. 3:47-71.

McCalla, D. R., D. J. Morre, and D. J. Osborn. 1962.
The metabolism of a kinin, benzyladenine. Biochem.
et Biophys. Acta 55:522-528.

Miller, C. O. 1961. Kinetin and related compounds in
plant growth. Ann. Rev. Plant Physiol. 12:395-408.

Miller, C. O. 1961. A kinetin-like compound in maize.
Proc. Natl. Acad. Sci. 47:170-174.

Mothes, K., and L. Engelbrechth. 1961. Kinetin-induced
directed transport of substances in excised leaves
in the dark. Phytochem. 1:58-62.

Parker, G. W., and D. S. Letham. 1973. Regulators of
cell division in plant tissues 16. Metabolism of
zeatin by radish cotyledons and hypocotyls. Planta
114:199-218.

Phillips, I. D. J. 1971. Introduction to the bio-
chemistry and physiology of plant growth hormones.
McGraw-Hill Book Co., New York 173 pp.

Pratt, H. K., and J. D. Goeschl. 1969. Physiological
roles of ethylene in plants. Ann. Rev. Plant Physiol.
20:541-584.

Quinlan, J. D., and R. J. Weaver. 1969. Influence of
benzyladenine, leaf darkening, and ringing on move-
ment of l4C-labeled assimilates into expanded leaves
of Vitis vinefera L. Plant Physiol. 44:1247-1252.

 

Smith, 0. E., and C. E. Palmer. 1970. Cytokinin-
induced tuber formation on stolons of Solanum
tuberosum. Physiol. Plant. 23:599-606.

 

56

12.

13.

14.

15.

57

Steward, F. C., and E. M. Shantz. 1959. The chemical
regulation of growth: Some substances and extracts
which induce growth and morphogenesis. Ann. Rev.
Plant Physiol. 10:379-404.

Sugiura, M., K. Umemira, and Y. Oota. 1962. The effect
of kinetin on protein levels of tobacco leaf discs.
Physiol. Plant. 15:457-464.

Taylor, P. A., T. Kosuge, and J. E. DeVay. 1974. Com-
pounds associated with cytokinin activity in fruitlets
and tracheal fluid of Gossypium hirsutum. Physiol.
Plant. 30:119-124.

 

Wang, D. 1961. The nature of starch accumulation at the
rust-infection site in leaves of pinto bean plant.
Can. J. Bot. 39:1595-1604.

CHAPTER 4

SUMMARY AND CONCLUSIONS

A series of enzymes, distinguished from one another by
their activity pH optima, were extracted from the endosperm
of developing corn seeds. These enzymes have similar sub-
strate specificities, being able to hydrolyze a-N-benzoyl-
d1 -Arginine-p—nitroanilide, but are ineffective against zein,
bovine albumin, wheat gliadin, and Azocoll. At least some are
inhibited by an extract containing the endogenous trypsin in-
hibitor. Because of their specificity, it is postulated that
these peptidases are involved in normal protein turnover
rather than hydrolysis of storage proteins. Since the reaction
between the peptidase and the trypsin inhibitor destroys the
active site--thus the activity--of the inhibitor, it is likely
that the two are physically separated in the intact endosperm.
A synthesis of appropriate literature indicates that, upon
germination, the trypsin inhibitor is lost from the endosperm
at a rate roughly equivalent to that of other protein frac-
tions. In view of this, and the fact that no role for the
trypsin inhibitor-peptidase relationship was obvious from
this study, the author suggests that the trypsin inhibitor

behaves as storage protein in the endosperm of corn.

58

59

Using linear regression analysis and a comparison of
developmental patterns, it was determined that although the
Opaque-2 variety was higher in both trypsin inhibitor and
lysine than its normal isoline, the relationship was inci-
dental, not causal. The data suggested that the higher trypsin
inhibitor was due to the higher albumin content of the Opaque-
2 line. Since more lysine is contributed by the glutelin
fraction than by the albumin fraction in Opaque-2 corn, it
may be possible to produce a high-lysine, low trypsin inhibi-
tor line by selecting for low albumin. The Opaque-2 line was
also higher in moisture and peptidase than its normal counter-
part.

Injections of benzyladenine influenced several para-
meters within the developing endosperm. The lines did not
always respond to the same degree, or for the same duration,
but the effect of the treatment was similar in every case.
Benzyladenine accelerated the developmental trend typical of
the variety. Different parameters responded to different
treatment times, but no parameter responded to both treatment
times. This indicated that there may have been different
"critical periods" for the translation of genetic information.
The data presented here strongly suggests that sequential
events within the developing endosperm are controlled by en-

dogenous plant growth substances.

APPENDICES

APPENDIX A

DETERMINING THE PROTEIN CONTENT OF CORN ENDOSPERM FLOUR

a)

b)

C)

d)

e)

f)

g)

h)

i)

Flour (50 to 100 mg) is weighed into 100 ml Kjeldahl
flasks marked at 100 ml.

2 ml concentrated sulfuric acid and 2 selenized
boiling chips are added.

The mixture is digested until colorless.

Cool. 0.5 ml concentrated hydrogen peroxide is
added and the mixture is digested for another 10
minutes.

After cooling, about 60 mls of 0.33 N NaOH is added
with vigorous mixing. The flask is then brought to
volume with 0.33 N NaOH.

The solution is allowed to stand at room temperature
for 15 minutes, then 1 m1 of each solution is re-
moved to a clean 100 m1 volumetric flask.

2 ml of H2S04 are added with mixing, then 10 m1 of
water is added.

When the mixture has cooled, 10 ml of 40% NaOH is
added and the flask is shaken.

2 ml of Sigma Ammonia Color Reagent is added. The

solution is mixed and immediately diluted to volume

 

60

61

with water.
NOTE: The final solution contains very small
amounts of N. For precise results glassware must
be meticulously clean and all water added should
be distilled and deionized.
j) Absorbance is read at 450 mu.after 15 minutes.
Nitrogen content can be determined from a standard
curve (NH4SO4 was used in this study). Using a factor of

6.25 to convert mg NH4 to mg protein, results accurate to

f 3% were routinely obtained.

APPENDIX B

DETERMINING THE PEPTIDASE AND TRYPSIN INHIBITOR

(TI) CONTENT OF CORN ENDOSPERM MEAL WITH

a-N-Benzoyl-dl-Arginine-p-Nitro-anilide (BAPA)

A. Reagents

1.

Tris buffer (0.05 M, containing 0.02 M CaClz)

6.05 gm tris-(hydroxymethyl) aminomethane (Sigma)

2
2. H20.

The above chemicals are added to 900 ml distilled

2.94 gm CaCl

water and the pH adjusted with 1 M HCl or 1 M
NaOH and brought to l L.
BAPA solution

30 mg BAPA HCl (Sigma) are dissolved completely per

 

ml of dimethylsulfoxide (DMSO) and diluted to 100

ml with tris buffer pre-warmed to 37 C. This so-

lution breaks down within a few hours.

Trypsin solution

5 mg trypsin (2 x crystallized, Worthington Biochem.

corp.) is dissolved in 100 ml 0.001 M HCl. This

solution can be stored for several days in the cold.

Extraction of the peptidase

a) Freeze-dried flour is weighed into centrifuge
tubes.

b) Distilled water is added. (One gm in 10 ml was

62

63

used to extract at pH 5.0 for this study, and
.25 gm in 10 was used to extract at pH 9.0).

c) The tubes are agitated until an homogenous
suspension is attained.

d) The pH is adjusted with 1 M NaOH or 1M HCl.
Before reading pH, the suspension must be
thoroughly agitated after each addition of
base or acid.

e) The solution is extracted for 2 hours at 4 C.

f) After centrifugation at 17,300 x G (O C) for
15 minutes the supernatant fluid is decanted,
diluted 1:1 with distilled water, and used as
a crude peptidase preparation.

NOTE: Corn endosperm flour contains both TI and

peptidase. The reaction of these enzymes destroys

the activity of the TI, thus increasing the activity
of the peptidase (see Appendix E). Care must be
taken to keep the solution cold before use. It
should be used immediately after dilution, as delays
may introduce variations in both TI and peptidase
activity.

Extraction of the trypsin inhibitor

a) Steps a through e above are repeated.

b) After centrifugation as above, ELL of the crude
supernatant fluid is removed to appropriate

glassware and held at 80 C for 10 min.

64

c) The solution is then placed in clean centrifuge
tubes and centrifuged for 10 minutes at 12,100
x G (O C).
d) The supernatant fluid is decanted and diluted
to a concentration affording 50% inhibition.
NOTE: Dilutions of the TI solution are made after
the second centrifugation because the more concen-
trated solution tends to form a firm, distinct
pellet, thus facilitating the handling of a clean
supernatant fluid. Excess protein and lipid in the
assay solution interferes with precise measurement

of the nitroanilide release.

B. Procedure

Trypsin standard curve

a) 0.2 to 1.0 m1 of the stock trypsin solution is
pipetted to a triplicate set of test tubes, and
the volume made up to 2 ml with distilled water.
To one set of the triplicate, add 1 m1 of 30%
acetic acid. These tubes will serve as blanks.

b) The tubes are then placed in a water bath at 37 C.
The reaction is begun by adding 7 m1 of the BAPA
solution, pre-warmed to 37 C.

c) After 10 minutes the reaction is stopped with the
addition of 1 m1 of 30% acetic acid.

d) The mixtures are allowed to equilibrate for at

least 20 minutes at room temperature, and the

65

absorbance of each is read at 410 mu against
the appropriate blank. The values of the 2
reaction mixtures are averaged.
2. Peptidase assay

a) Three test tubes are used for each sample, at
each dilution. 0.5 ml or 1.0 m1 of the crude
peptidase preparation is pipetted into each of
the 3 tubes. The volume is brought to 2 ml
with distilled water.

b) To 1 tube 1 ml of 30% acetic acid is added.
This solution serves as a blank.

c) The tubes are then placed in the 37 C water
bath.

d) After 2 minutes the reaction is begun by adding
7 ml of the warm BAPA buffered solution.

e) Exactly 10 minutes later the reaction is termi-
nated with 1 m1 of 30% acetic acid.

NOTE: Since small variations in the concentration

of the BAPA solution and time of assay are probable,

one sample of a trypsin solution of known activity

is run with each group of peptidase assays. One

such value is chosen as a standard and all unknowns

are then multiplied by the appropriate factor to

provide the change in Optical Density (A O. D.)

which is due to peptidase activity. In this manner

day-to-day variations are minimized.

66

Trypsin inhibitor assay

a)

b)

C)

d)

e)

0.5 or 1.0 ml of the cold TI solution is added
to a triplicate set of test tubes.

Distilled water is added to 1 ml.

1.0 ml of trypsin solution is added to each
tube.

Blanks receive 1 ml of 30% acetic acid.

The reaction is begun without pre-warming and

terminated as above.

Expression of activity

Percent inhibition is easily calculated from
the differences in absorbances between unknown
and the stock trypsin solutions. Since 50 mg
trypsin were added, 50 x percent inhibition/
100 = mg trypsin inhibited per 10 ml of the
described solution, under the described condi-

tions.

APPENDIX C

TABLE C-l.

PERCENT VARIATION

BETWEEN REPLICATIONS

AS AFFECTED BY TIME AND VARIETY.a

DAYS AFTER POLLINATION

 

 

Parameter 20 27 34 41
Total protein (Opaque-2) 14.8 13.0 21.7 18.
Total protein (normal) 30.6 15.0 14.6 19.
Water-soluble protein (Opaque-2) 62.5 18.0 77.4 24.
Water-soluble protein (normal) 42.3 26.4 88.0 66.
Trypsin inhibitor (Opaque-2) ---- 31.9 17.9 6.
Trypsin inhibitor (normal) —--— 35.00 27.0 14.
Peptidase pH 9.0 (Opaque-2) 10.1 4.0 52.7 47.
Peptidase pH 9.0 (normal) 25.1 34.4 24.2 69.
Peptidase pH 5.0 (Opaque-2) 42.5 17.1 58.1 29.
Peptidase pH 5.0 (normal) 29.7 13.9 11.7 70.
Dye-binding capacity (Opaque—2) 3.0 5.9 3.9 22.
Dye-binding capacity (normal) 5.5 9.1 7.2 10.

a . .
Variations as percent of mean value.

67

APPENDIX D

TABLE D-l. THE pH OF 1 gm OF ENDOSPERM
FLOUR AND 10 ml DISTILLED WATER.a

DAYS AFTER POLLINATION

 

13 20 27 34 41

lo
2
IO
2
lo
12
lo
2
0

Rep. 1 5.89 5.92 6.06 5.82 6.67 6.71 7.1 6.84 6.88

Rep. 2 5.9 5.95 6.67 6.71 7.04 6.91 6.92
Rep. 3 5.94 6.03 6.77 6.66 7.05 6.91 6.78
Rep. 4 5.96 6.13 6.79 6.78 7.06 6.92 6.75
Rep. 5 5.96 6.13 6.79 6.78 7.06 6.92 6.75
Rep. 6 6.13 5.97 6.75 6.74 7.06 6.94 6.8
Rep. 7 6.1 6.25 6.76 6.76 7.06 6.92 6.78
Rep. 8 6.06 5.94 6.77 6.7 7.07 6.96

aValues are those obtained from untreated
plants.

bO Opaque-2 N normal

68

69

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APPENDIX F

Figure F-l Amount albumin extracted as a function of

the pH of the extracting solution.

70

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mg soluble protein/gm flour

 

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APPENDIX G

Figure G-l Peptidase activity when assayed at pH 9.0,

as a function of the pH of the extracting solution.

72

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APPENDIX H

Figure H-l Trypsin inhibition using corn endosperm
trypsin inhibitor. The broken line demonstrates inhibition
using extract from the normal endosperm variety. The solid
line demonstrates results of extract from the Opaque-2

variety of the same age.

74

Percent inhibition

90

80

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60

50

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