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This is to certify that the

dissertation entitled

PROTEINASES IN SOFT WINTER WHEAT DURING
MATURATION, PREHARVEST SPROUTING AND GERMINATION

presented by

Christine Joy Bergman

has been accepted towards fulfillment
of the requirements for

Ph. D. degree in Food Science

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Date Sept. 9, 1994

 

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TO AVOID FINES Mum on or before date duo.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

PROTEINASES IN SOFT WINTER WHEAT DURING
MATURATION, PREHARVEST SPROUTING AND GERMINATION

By

Christine Joy Bergman

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Food Science and Human Nutrition

1994

ABSTRACT

PROTEINASES IN SOFT WINTER WHEAT DURING
MATURATION, PREHARVEST SPROUTING AND GERMINATION

By

Christine Joy Bergman

Along with alpha-amylase activity, proteinase activity has been linked with
the detrimental effects of preharvest sprouting (PHS) on wheat product quality.
However, many questions remain regarding these enzymes. Those addressed in
this study are: is proteinases activity affected by environment or PHS; is
proteinase activity correlated with alpha-amylase activity or rate of germination;
what classes of proteinase are present in maturing and germinating soft winter
wheat (SW); and how do proteinases affect storage proteins during
germination. Five red and four white SWW were grown in a greenhouse (GHG)
and in Dansville, Michigan, in 1992. Samples were harvested at 21, 28, 35 and
42 days post anthesis. Additionally, kernels of GHG Augusta and Hillsdale were
germinated for five days. Throughout kernel maturation, proteinase activity was
significantly higher in the field-grown grain (FGG) compared to the GHG. At

harvest maturity, proteinase and alpha-amylase activities were positively

correlated (r = 0.669, p < 0.05) in the F66. Also, germination index was
correlated with proteinase (r = 0.553, p < 0.05) and alpha-amylase activities (r =
0.622, p < 0.05). Crude extracts from GHG Augusta contained cysteine and
aspartic proteinase activity during kernel maturation. As a percentage of total
proteinase activity, cysteine proteolytic activity decreased as kernels matured,
while the aspartic activity increased. During germination, cysteine proteinase
activity increased, while aspartic proteinase activity decreased until the second
day of germination. These trends then reversed. After one day of germination,
serine and metallo-proteinase activity began to increase and peaked after four
days. Ten bands of proteinase activity were visualized using native
polyacrylamide gel electrophoresis with copolymerized gelatin and crude
extracts from quiescent and germinated Augusta kernels. Two bands were
present in quiescent grain at near neutral pH, suggesting that they contain
serine or metallo-proteinase. The other eight bands had acidic pH optima and
appeared after germination had commenced, indicating that they contain
aspartic or cysteine proteinase. Glutenin subunits in germinated kernels

appeared to be hydrolyzed by proteinases earlier than the gliadin proteins.

Copyright by

CHRISTINE JOY BERGMAN

1994

DEDICATION

I can do all things through Christ who strengthens me (Phil. 4:13)

I dedicate the completing of this dissertation to my Mom, Dad, my sister
Lori, brother Jeff, Grandmas, Beulah and Olga, and Grandpa Oliver, whom I love

deany.

ACKNOWLEDGMENTS

"Life is like a box of chocolates. You never know what you’re gonna get."
(Forest Gump’s Mother)

The following people played various roles in helping me complete this
dissertation: Daisy Gualberto, Lorraine Bergman, Karen Kane, Janice Harte, Bill
Haines, Nirmal Sinha, Lee Bird, Pedro Maligo, Debi Klein, Jenny Bond, Patrick
Finney, Arti Arora, Rick Bernsten, Eileen Van Tassel, Kitty O’Neil, Kati Loeffler,
Luis Rayas, Scott Worthington, Sandy Daubermier, Dave Glenn, Kay Kresl,
Cynthia Merriman and Jessalin Faulkner. A few of those mentioned above
provided me technical help, but all offered thought provoking discussions and

words of encouragement. I extend heartfelt thanks to all of them.

The financial assistance provided, in the form of fellowships and
scholarships, by the Institute of Food Technologists, the American Association of
Cereal Chemists, Michigan State University’s Graduate School and the US.

Government’s Title IV Foreign Language Program is acknowledged.

Thanks also go to the following professors for serving on my committee
and reviewing this dissertation: Jerry Cash, Perry Ng, Rick Ward and Mary Ellen
Zabik. My gratitude is especially offered to Rick Ward for providing the wheat

samples for this project, plus the field and greenhouse space.

vi

TABLE OF CONTENTS

LIST OF TABLES .............................................................................................. xii
LIST OF FIGURES ............................................................................................ xiii
LIST OF ABBREVIATIONS ............................................................................... xvi
CHAPTER ONE: INTRODUCTION ...................................................................... 1
CHAPTER TWO: LITERATURE REVIEW ........................................................... 4
INTRODUCTION .................................................................................................. 4
PREHARVEST SPROUTING or: WHEAT ....................................................................... 4
Geographic Occurrence and Impact ................................................................. 4
Effect of Preharvest Sprouting on Cereal Product Quality ................................ 5
~Hard Wheat Products .................................................................................... 5
Soft Wheat Products ...................................................................................... 9
Dormancy ........................................................................................................ 12
Physical Factors .......................................................................................... 12
Chemical Inhibitors and Promoters .............................................................. 14
Environmental Factors ................................................................................. 16
Preharvest Sprouting Methodology ................................................................. 16
Quantifying and Studying Preharvest Sprouting .......................................... 16

vii

Characterizing Resistance to Preharvest Sprouting .................................... 25

PHYSIOLOGY OF GERMINATION ............................................................................... 29
Grain Maturation .............................................................................................. 29
Water Imbibition .............................................................................................. 30

BIOCHEMISTRY OF GERMINATION ............................................................................ 31
Protein-Degrading Enzymes ............................................................................ 31

Proteinase Biochemistry .............................................................................. 32
Aspartic Proteinases ................................................................................ 32
Cysteine Proteinases ............................................................................... 34
Metallo-Proteinases ................................................................................. 35
Serine Proteinases ................................................................................... 36

Proteinases in Wheat .................................................................................. 37

Proteinases Effect on Wheat Proteins ......................................................... 39

Proteinases Effect on Wheat Product Quality ............................................. 41

Other Enzymes ................................................................................................ 42
REFERENCES ................................................................................................... 43

CHAPTER THREE: PROTEINASE ACTIVITY IN SOFT WINTER WHEAT

DURING MATURATION AND PREHARVEST SPROUTING ............................. 55
ABSTRACT ......................................................................................................... 55
INTRODUCTION ................................................................................................ 56
MATERIALS AND METHODS ............................................................................ 58

Materials .......................................................................................................... 58

viii

Wheat Samples ........................................................................................... 58

Chemicals .................................................................................................... 58
Methods .......................................................................................................... 58
Wheat Growing Conditions and Harvest ..................................................... 58

F ieId-Grown Wheat .................................................................................. 58
Greenhouse-Grown Wheat ...................................................................... 59
Sample Preparation ..................................................................................... 59
Physical Methods ........................................................................................ 61
Moisture ................................................................................................... 61
Germination Index .................................................................................... 61
Chemical Methods ....................................................................................... 62
Protein Content ........................................................................................ 62
Alpha-Amylase Activity ............................................................................. 62
Proteinase Activity ................................................................................... 62
Proteinase Inhibitors ................................................................................ 63
Statistical Analysis ....................................................................................... 64
RESULTS ........................................................................................................... 66
Inhibitor Studies ............................................................................................... 66
Enzyme Activity Dun'ng Maturation .................................................................. 66
Field Environmental Conditions ....................................................................... 77
Field-Weathered Grain .................................................................................... 77
Greenhouse-Grown Grain ............................................................................... 80

DISCUSSION .......................................................................... ‘ ............................ 80

REFERENCES ................................................................................................... 88

CHAPTER FOUR: PROTEINASES IN SOFT WINTER WHEAT DURING

GERMINATION AND THEIR EFFECT ON STORAGE PROTEINS ................... 91
ABSTRACT ......................................................................................................... 91
INTRODUCTION ................................................................................................ 92
MATERIAL AND METHODS ............................................................................... 93
Materials .......................................................................................................... 93
Wheat Samples ........................................................................................... 93
Chemicals .................................................................................................... 94
Methods .......................................................................................................... 94
Wheat Growing Conditions and Harvest ..................................................... 94
Sample Preparation ..................................................................................... 94
Controlled Germination ............................................................................ 94
Milling ....................................................................................................... 95
Germination Index ....................................................................................... 95
Proteinase Activity ....................................................................................... 95

Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

of Glutenins ................................................................................................. 96
Acid Polyacrylamide Gel Electrophoresis (PAGE) of Gliadins .................... 96
Proteinase Characterization ........................................................................ 96

Enzyme Extraction ................................................................................... 96

Native PAGE with Copolymerized Substrate ........................................... 96

Glutenin Preparation ................................................................................ 97
Proteinase Inhibitor Studies ..................................................................... 97
Statistical Analysis ....................................................................................... 97
RESULTS ........................................................................................................... 98
Germination Index ........................................................................................... 98
Proteinase Activity ........................................................................................... 98
Visualization of Proteinase Activity .................................................................. 98
Inhibitor Studies ............................................................................................. 103
Proteinase Effect on Storage Proteins .......................................................... 111
DISCUSSION .................................................................................................... 116
REFERENCES ................................................................................................. 127
CHAPTER FIVE: CONCLUSIONS ................................................................... 130
CHAPTER SIX: FUTURE WORK ..................................................................... 133
APPENDIX A .................................................................................................... 135
APPENDIX B .................................................................................................... 136
APPENDIX C .................................................................................................... 147

xi

LIST OF TABLES

TABLE I. TEMPERATURE AND PRECIPITATION IN THE VICINITY OF DANSVILLE, MICHIGAN,
DURING THE SUMMER OF 1992 ....................................................................... 60
TABLE II. CONDITIONS FOR PROTEINASE INHIBITOR STUDIES .................................... 65
TABLE III. CORRELATION ANALYSIS OF MATURING SOFT WINTER WHEAT ENZYME
ACTIVITIES WITH DPA AND PROTEIN CONTENT ................................................ 78
TABLE IV. CORRELATION ANALYSIS OF ENZYME ACTIVITIES WITH GERMINATION INDEX
AND PROTEIN CONTENT OF MATURE SOFT WINTER WHEAT GROWN IN DANSVILLE,
MICHIGAN, IN 1992 AND HARVESTED AT 42 DPA ............................................. 79
TABLE V. ALPHA-AMYLASE AND PROTEINASE ACTIVITY IN SOFT WINTER WHEAT
CULTIVARS GROWN IN DANSVILLE, MICHIGAN, IN 1992 AND HARVESTED AT 42

DPA ............................................................................................................. 81

xii

LIST OF FIGURES

FIGURE. 1. GRAPH OF RELATIVE PROTEINASE ACTIVITY OF THE CULTIVAR AUGUSTA
GROWN IN A GREENHOUSE AND HARVESTED AT 21 , 28, 35, AND 42 DAYS POST
ANTHESIS, WITH AND WITHOUT PEPSTATIN A. ................................................... 67

FIGURE. 2. GRAPH OF RELATIVE PROTEINASE ACTIVITY OF THE CULTIVAR AUGUSTA
GROWN IN A GREENHOUSE AND HARVESTED AT 21 , 28, 35, AND 42 DAYS POST
ANTHESIS, WITH AND WITHOUT IODOACETIC ACID ........................ ~ ...................... 68

FIGURE. 3. GRAPH OF RELATIVE PROTEINASE ACTIVITY OF THE CULTIVAR AUGUSTA
GROWN IN GREENHOUSE AND HARVESTED AT 21, 28, 35, AND 42 DAYS POST
ANTHESIS, WITH AND WITHOUT N-ETHYLMALEIMIDE ........................................... 69

FIGURE. 4. GRAPH OF RELATIVE PROTEINASE ACTIVITY OF THE CULTIVAR AUGUSTA
GROWN IN A GREENHOUSE AND HARVESTED AT 21 , 28, 35, AND 42 DAYS POST
ANTHESIS, WITH AND WITHOUT P-CHLOROMERCURIPHENYL SULFONIC ACID ........ 70

FIGURE. 5. GRAPH OF RELATIVE PROTEINASE ACTIVITY OF THE CULTIVAR AUGUSTA
GROWN IN A GREENHOUSE AND HARVESTED AT 21 , 28, 35, AND 42 DAYS POST

ANTHESIS, WITH AND WITHOUT 3-CARBOXYL-2, 3-L-TRANS-EPOXYPROPYL-

LEUCYLAMIDO (4-GUANIDINo) BUTANE (E-64) .................................................. 71

xiii

FIGURE. 6. GRAPH OF RELATIVE PROTEINASE ACTIVITY OF THE CULTIVAR AUGUSTA
GROWN IN A GREENHOUSE AND HARVESTED AT 21 , 28, 35, AND 42 DAYS POST
ANTHESIS, WITH AND WITHOUT 1, 10 PHENANTHROLINE .................................... 72

FIGURE. 7. GRAPH OF RELATIVE PROTEINASE ACTIVITY OF THE CULTIVAR AUGUSTA
GROWN IN A GREENHOUSE AND HARVESTED AT 21 , 28, 35, AND 42 DAYS POST
ANTHESIS, WITH AND WITHOUT 3,4 DICHLOROISOCOUMARIN .............................. 73

FIGURE. 8. GRAPH OF RELATIVE PROTEINASE ACTIVITY IN SOFT WINTER WHEAT GROWN
IN A GREENHOUSE AND IN DANSVILLE, MICHIGAN, IN 1992 ................................ 75

FIGURE. 9. GRAPH OF ALPHA-AMYLASE ACTIVITY IN SOFT WINTER WHEAT GROWN IN A
GREENHOUSE AND IN DANSVILLE, MICHIGAN, IN 1992 ........................................ 76

FIGURE. 10. GRAPH OF THE RELATIVE PROTEINASE ACTIVITY OF THE CULTIVARS
AUGUSTA AND HILLSDALE GROWN IN A GREENHOUSE AND EXPOSED To
CONDITIONS CONDUSIVE To GERMINATION IN A LABORATORY ............................. 99

FIGURE. 11. PAGE WITH COPOLYMERIZED GELATIN OF SOFT WHEAT CRUDE EXTRACTS
PREPARED FROM GRAIN AT DIFFERENT GERMINATION STAGES ......................... 100

FIGURE 12. GRAPH OF RELATIVE PROTEINASE ACTIVITY OF THE CULTIVAR AUGUSTA,
GROWN IN A GREENHOUSE, WITH AND WITHOUT PEPSTATIN A .......................... 104

FIGURE. 13. LINE GRAPH OF RELATIVE PROTEINASE ACTIVITY OF THE CULTIVAR
AUGUSTA, GROWN IN A GREENHOUSE, WITH AND WITHOUT IODOACETIC ACID.. . 105

FIGURE. 14. GRAPH OF RELATIVE PROTEINASE ACTIVITY OF THE CULTIVAR AUGUSTA,
GROWN IN A GREENHOUSE, WITH AND WITHOUT 3-CARBOXYL-2, 3-L-TRANS-EPOXY

PROPYL-LEUCYLAMIDO (4-GUANIDINo) BUTANE (E-64) .................................... 106

xiv

FIGURE. 15. GRAPH OF RELATIVE PROTEINASE ACTIVITY OF THE CULTIVAR AUGUSTA,
GROWN IN A GREENHOUSE, WITH AND WITHOUT N-ETHYLMALEIMIDE ................ 107
FIGURE. 16. GRAPH OF RELATIVE PROTEINASE ACTIVITY OF THE CULTIVAR AUGUSTA,
GROWN IN A GREENHOUSE, WITH AND WITHOUT P-CHLOROMERCURIPHENYL
SULFONIC ACID ............................................................................................ 108
FIGURE. 17. GRAPH OF RELATIVE PROTEINASE ACTIVITY OF THE CULTIVAR AUGUSTA,
GROWN IN A GREENHOUSE, WITH AND WITHOUT 1, 10 PHENANTHROLINE ......... 109
FIGURE. 18. GRAPH OF RELATIVE PROTEINASE ACTIVITY OF THE CULTIVAR AUGUSTA,
GROWN IN A GREENHOUSE, WITH AND WITHOUT 3,4 DICHLOROISOCOUMARIN .. 1 10
FIGURE. 19. SDS-PAGE OF REDUCED HMW-GLUTENINS FROM THE CULTIVARS
AUGUSTA AND HILLSDALE AT VARIOUS STAGES OF GERMINATION ..................... 1 12
FIGURE. 20. SOS-PAGE OF UNREDUCED HMW-GLUTENINS FROM THE CULTIVARS
AUGUSTA AND HILLSDALE AT VARIOUS STAGES OF GERMINATION ..................... 1 14
FIGURE. 21. ACID-PAGE OF GLIADIN PROTEINS FROM THE CULTIVARS AUGUSTA AND

HILLSDALE AT VARIOUS STAGES OF GERMINATION ........................................... I I7

XV

LIST OF ABBREVIATIONS

ABA abSCiSic acid
A-CL Augusta control
A-1 D, A-2 D ......... A-5 D Augusta kernels germinated for one

day, two days and subsequent

days up to five

CMPS p-Chloromercuriphenyl sulfonic acid

DAN diazoacetyl amino acid esters in the

presence of cupric ion

3, 4 DIC 3, 4 dichloroisocoumarin

DMSO dimethyl sulfoxide

DFP diisopropyl fluorophosphate

DPA days post anthesis

DTT dithiothreitol

E-64 3-carboxyl-2, 3-L-trans-epoxypropyl-

leucylamido (4-guanidino) butane
EDTA ethylenediaminetetraacetic acid

ELISA enzyme linked immunosorbent assay

xvi

FDB

FGG
GA
GHG
H-Cl

H-‘I D, H-2D ........ H-5 D

HM

HMW

kDa

LMHAA

2-ME
MUH
MW

PAGE

1, 10 PA
PHS

PM
PMSF

RH

fluorescein dibutyrate

field-grown grain
gibberellic acid
greenhouse-grown grain

Hillsdale control

Hillsdale kernels germinated for one
day, two days and subsequent

days up to five

harvest maturity

high molecular weight

kflodanon

late maturity high alpha-amylase

2-mercaptoethanol
4-methylumbelliferyl
molecular weight

polyacrylamide gel electrophoresis

1, 10 phenanthroline
preharvest sprouting
physiological maturity
phenylmethane sulfonylfluoride

relative humidity

xvii

SDS-PAGE sodium dodecyl sulfate-PAGE
TCA trichloroacetic acid

TEMED N, N, N', N'-Tetramethylethylenediamine

xviii

CHAPTER ONE

INTRODUCTION

Preharvest sprouting (PHS) damage, which occurs when cereal grains
with limited dormancy are exposed to precipitation just prior to harvest, is of
concern world wide due to the economic losses which result from decreased

yield, loss of seed viability, and poor flour quality (Tipples et al 1966).

Preharvest sprouting consists of root and shoot growth, as well as starch
and protein modification, from a cascade of events involving hormones and
enzymes. These changes are thought to be regulated at the molecular level, as
increased mRNA synthesis occurs within 24 h of wheat kernel imbibition
(Weidner and Kulka 1980). Concurrently, gibberellic acid travels from the
embryo to the aleurone layer triggering reserve mobilization through several
enzyme responses: increased de novo synthesis, secretion, and activation of
preexisting forms. Increased activity has been reported for carbohydrate,
protein, and lipid hydrolytic enzymes during sprouting, with the most
concentrated research efforts being on delineating the role of alpha-amylase

(Ashford and Gubler 1984).

Proteases in wheat have been studied Since the early part of this century
(Balls and Hate 1936). However, much about these enzymes still eludes us.
Such as: what classes of proteinases are present during the various stages of a
kernel's life cycle; where are they located in the kernel; what are their substrate
specificities; what are their temperature and pH Optima; and where are the

genes located which code for them?

2

Proteinase activity has been reported to increase approximately 20-fold
after three to five days of germination. These enzymes are known to have the
ability to hydrolyze gliadins and glutenins, and thus, in addition to alpha-amylase
conceivably may play a role in the effects of PHS on product quality (Preston

1978, Okamota et al 1981, Kruger 1990).

The study of proteinases, Specifically in soft wheat, is of interest, in part,
because of the potential role they may play in the end-product quality of
sprouted grain. Although increased alpha-amylase activity in sprouted grain has
been thought to be of greater consequence than other enzymes present, a few
studies have linked proteinase activity with a change in product quality (Lorenz
et al 1983, Edwards et al 1989). Also, greater protease activity compared to
alpha-amylase activity, in rain-damaged grain, has been reported to be carried

over from grain to flour after milling (Edwards et al 1989).

The focus of this study was to investigate several questions related to the
hypothesis that proteinase activity, in sprouted soft wheat, results in biochemical
Changes which modify end-product quality. These questions included: (1) what
Classes of proteinase are present in maturing soft winter wheat; (2) is proteinase
activity during kernel maturation affected by environment; (3) is proteinase
activity at harvest maturity correlated with alpha-amylase activity, or degree of
sprouting; and (4) does proteinase activity increase when grain sprouts prior to
harvest? In addition, this project sought to gain a "big-picture" perspective of the
proteinases in quiescent and germinating soft wheat, and to Characterize their

effects on soft wheat storage proteins.

3

REFERENCES

AShford, AB and Gubler, F. 1984. Mobilization of polysaccharide reserves from
endosperm. In: Seed Physiology; Germination and Reserve Mobilization.
II. D.R. Murray (ed.). Academic Press, Australia, Sydney, New York,
Toronto. pp.117-162.

Balls, A. K., and Hall, W.S. 1936. Proteolytic enzymes of flour. Cereal Chem.
13:54.

Edwards, R. A., Ross, A. S., Mares, D. J., Ellison, F. W. and Tomlinson, J. D.
1989. Enzymes from rain-damaged and laboratory-germinated wheat. I.
Effects on product quality. J. Cereal Sci. 10:157.

Kruger, J.E. 1990. Monitoring for sprout-damage in cereals and its implications
for end-product quality. Fifth International Symposium on Pre-Harvest
Sprouting in Cereals. K. Ringlund, E. Mosleth, and D. Mares (eds).
Westview Press, San Francisco. pp. 321-328.

Lorenz, K., Roewe-Smith, P., Kulp, K. and Bates, L. 1983. Preharvest sprouting
of winter wheat. ll. Amino acid composition and functionality of flour and
flour fractions. Cereal Chem. 60: 360.

Okamoto, K., Kitano, H. and Akazawa, T. 1981. Biosynthesis and excretion of
hydrolases in germinating cereal seeds. Plant Cell Physiol. 21:201.

Preston, K. R. 1978. Note on separation and partial purification of wheat
proteases by affinity Chromatography. Cereal Chem. 55:793.

Tipples, K.H., Kilborn, RH. and Bushuk, W. 1966. Effect of malt and sprouted
wheat in conventional and mechanical development breadmaking
processes. Cereal Sci. Today 11:362.

Weidner, S. and Kulka, K. 1980. Ribonucleic acids in the embryos of
germinating wheat grain of different ripeness. Acta. Soc. Bot. Pol. 49:423.

CHAPTER TWO
LITERATURE REVIEW

INTRODUCTION

Preharvest sprouting (PHS) is a phenomenum where physiologically ripe
cereal grains begin the germination process prior to being harvested. This
situation tends to occur when genotypes with limited dormancy are exposed to
steady rains followed by cool temperature, as opposed to short rains followed by
hot dry periods. Preharvest Sprouting consists of increased hydrolytic enzyme
activity and, as a result, grain which is less suitable for processing into food
products. Although PHS is defined as the germination of grain still in the plant
ear, for ease of experimentation and control of the“ environment, laboratory-

germinated material is often used to study the effects of PHS.

Preharvest Sprouting of Wheat
Geographic Occurrence and Impact

Sprouting damage is of concern world wide due to the economic losses
which result from decreased yield, loss of seed viability, and poor flour quality
(Tipples et al 1966). Areas of the world particularly susceptible to preharvest
sprouting are Argentina, Brazil, Chile, Eastern New Zealand, England, Northern
Europe, Kenya, South Africa, Zimbabwe, and the Canadian regions of
Saskatchewan and Manitoba. For example, in 1987 England realized a $114

Million loss due to winter wheat sprouting (Derera 1990). In the United States,

5

white wheat growing areas, including the Pacific Northwest (Finney et al 1980),
New York (Briggle 1980), and Kansas (McCrate et al 1981), are affected most
often. Michigan, primarily a soft white wheat producer, has also experienced
PHS, with an estimated 50-60% of its acreage containing sprouted grain in 1980

(Copeland et al 1980).

Effect of Preharvest Sprouting on Cereal Product Quality

Hard Wheat Products

Leavened bread, hard wheat's primary product, is consumed world wide.
For example, bread consumption in Hungry, France, United States, Brazil, and
Japan is 120, 67, 37, 19 and 10 kg/personlyear, respectively (Orth and
Shellenberger 1988). With the protein and kilocalorie needs of SO many
dependent on this one product plus the potential for economic losses, the effect

of PHS on hard wheat has received considerable attention.

Millers have concerns about sprouted grain not only because of its effect
on product quality, but also due to its potential for changes in milling quality.
Soaking and to a lesser extent sprouting have been reported to result in hard
wheat acquiring some of the milling traits of soft wheat (Lukow and Bushuk
1984a). That is, break-stage yields increased to the same extent that reduction
flours decreased with no Change in overall yield. Depending on the intended use

of the flour, millers may find this a benefit or a drawback.

For the baker, the first manifestation of sprouting is generally identified by
dough that is sticky and hard to handle (D'Appolonia 1983). However, with a
method that uses a short fermentation time, such as the Chorleywood process,

signs of sprouting may not be seen until the product reaches the slicing machine

6

(Chamberlain et al 1983). Loaves made from Sprout-damaged wheat can
become deformed as they travel through a slicer. The Slices become uneven,
stick together, and sticky dough builds up on the blades. In time, this build-up of
gummy material results in a loaf being ripped apart and Slicing is brought to a
halt. With several hours of production backed up at this point, the economic

losses can be substantial (Meredith and Pomeranz 1985).

Examination of the effect of sprouting on bread quality began with work by
Kozmin (1933), when the author reported compromised dough handling, crumb
color, grain, and texture. Later studies tend to agree that sprouting results in a
sticky dough, very dark crust, open grain, either a gray or yellowish crumb color,
and in extreme cases, collapsed loaves (Ibrahim and D'Appolonia 1979, Lorenz

et al 1983, Ariyama and Khan 1990).

The effect of sprouting on loaf volume is complex. Ranhotra et al (1977)
reported that at levels less than five percent, sprouting results in larger loaf
volumes; at higher levels, loaf volumes were said to decrease. Work by Lorenz
et al (1983) determined that bread volume increased at all levels of sprouting.
Ariyama and Khan (1990) found greater loaf volumes for laboratory sprouted
hard wheat. However, grain sprouted for 12 h produced loaves with larger
volumes than that sprouted for 40 h It has been suggested that sprouting
displays a concomitant rise in loaf volume, because the sugars hydrolyzed by
alpha-amylase provide superior levels of fermentable sugars and thus more C02

is produced (Lorenz et al 1983).

Suggestions have been made as to the amount of sprouting that can be
tolerated by a bakery (Ranhotra et al 1977). However, a study of commercial
bakeries in England concluded that tolerable levels of alpha-amylase activity,

using the Chorleywood process, varied according to the type of product being

produced and the equipment used. For example, one bakery's slicing operation
was successful until 20 Farrand units was reached while another began to have

problems at 10 Farrand units (Buchanan and Nicholas 1980).

Determining tolerable levels of Sprouting is thus a rather arduous task
considering the many variables possibly involved in the effect Of sprouting on
product quality: variety of wheat, type of product, formulation, processing time,
and equipment used. This issue is further complicated when it is realized that
the degree of Sprouting is generally reported based on alpha-amylase activity. If
a high percentage of a lot of grain were exposed to water prior to harvesting,
other germinative enzymes which may also affect product quality may or may not
be present in significant amounts. For example, proteolytic enzymes have been
reported to be involved in the effect of germination on leavened bread quality
(Kruger 1990). Thus, traditional methods of reporting PHS may not be able to
adequately predict product quality.

AS it is currently accepted that PHS is a detriment to leavened bread
quality, more recent work has focused on establishing the physicochemical
changes which result in the modified functional properties of wheat components.
The hope is that this type of work will lead not only to a better understanding of
sprouting, but also may determine specific applications where germinated grain

can be used without loss of quality.

Dough stickiness has, by tradition, been attributed to starch modification
during Sprouting. Evidence for this was not available until Lorenz et al (1983)
individually recombined starch and gluten from flour milled from sprout-damaged
grain with a control flour's starch or gluten. Stickiness occurred when starch from
sprouted grain was recombined with sound gluten. In contrast, dough produced

from Sprout-damaged gluten with a control starch demonstrated no stickiness.

8

Lorenz and Kulp (1981) demonstrated that laboratory-sprouted hard
wheat starch exhibits reduced swelling power, is more soluble, gelatinizes at a
lower temperature over a narrower temperature range, and has a lower falling

number and amylograph viscosities.

Four varieties of hard wheat, which contained from 2 to 80% field-
sprouted kernels, did not display swelling power to be a function of degree of
sprouting (Kulp et al 1983). Nor was PHS found to affect starch solubility of
samples that had undergone an enzyme inactivation step after extraction.
However, when enzyme inactivation was not performed, considerable increases
in solubility accompanied samples with greater levels of sprouting. The authors
suggested that, during the solubility measurement, alpha-amylase on the starch
granule surfaces was exposed to temperatures ideal for this enzyme's activity.
Thus, the difference in solubilities was due to hydrolysis that occurred after
initiation of the solubility measurement. Thus, it appears that alpha-amylase
damage does not occur in situ, but rather after the dough has been hydrated. As
with laboratory sprouting, field sprouting was associated with slight increases in
water binding capacity, with no difference noted between enzyme inactivated
and noninactivated starch. ln opposition to laboratory-sprouted grain, field-

sprouted wheat displayed no change in gelatinization temperature ranges.

Starch granules from wheat varieties containing 0, 40, and 78%
germinated grain displayed no Sign of damage from higher amylase activity,
using scanning electron microscopy and x-ray diffraction patterns (Kulp et al
1983). This was contrary to findings by Dronzek et al (1972) and Bean et al
(1974), who determined that laboratory Sprouting resulted in extensive starch
granule damage. Kulp et al (1983) suggested that the differences between

starch granules and their functionality, in field- versus laboratory-sprouted

9

wheat, is because field sprouting does not achieve the same level of starch
degradation that laboratory-sprouted grain does. That is, conditions for
germination (temperature and moisture levels) are not held constant, and thus,

are less than ideal.

Along with increases in carbohydrate-degrading enzymes, germination
also results in greater protease activity. As a result, there has been some
question as to the role protein modification may play in the detrimental effects of
Sprouting on product quality. This topic will be covered in detail under the

proteinase section.

Soft Wheat Products

Studies have established that sprouting of hard wheat results in sticky
dough and bread with inferior crumb and coarse texture (Kulp and Lorenz 1983).
However, the PHS potential of soft wheats and its effects on soft wheat product
quality has, in comparison, received limited attention. Perhaps in part due to the
large variety of products soft wheat is used for or because soft wheats, in

general, have received less research attention than hard wheats.

Triticum aestivum varieties which have soft-textured kernels are known as
soft wheats. Generally lower in protein than hard wheats, soft wheat cultivars are
known as the wheat of choice for a large variety of products. Soft wheat product
categories include: biscuits, some breakfast cereals, breadings, cakes, cookies,
crackers, doughnuts, flat breads, oriental noodles, pancakes, pastries (including

pie crusts), pretzels, some snack foods, thickeners, waffles, and wafers.

Although the effect of sprouting on product quality has primarily focused
on hard wheat leavened bread, a few studies have examined its effect on the

quality of soft wheat products. A soft white wheat cultivar, which was laboratory

70

sprouted for four days, was reported to have flour extraction rates within the
commercial range for soft wheat applications (Lorenz and Valvano 1981).
Sorrells et al (1989) subjected sprouting resistant and susceptible soft wheat
varieties to controlled field sprouting. Sprouting susceptible cultivars yielded the
same amount of flour as the control while those resistant to sprouting had lower

flour yields.

Lorenz and Valvano (1981) found improved spread ratios and subjective
grain score measurements from cookies made using a laboratory sprouted soft
wheat. The cookies were darker in color apparently due to increased sugar
levels, which provided favorable conditions for the maillard reaction. Sprouting
had no effect on the cookies flavor score, but a sensory panel did give the
sprouted products lower scores for texture and appearance. Sorrells et al (1989)
found no difference between a control and field sprouted soft wheat cookie

spread and appearance.

From these studies it is difficult to draw conclusions about the effect of
sprouting on cookie quality because different sprouting methods were used, and
the methods used to report degree of sprouting cannot be compared. Lorenz and
Valvano (1981) reported falling number while Sorrells et al (1989) determined
percent germination and alpha-amylase activity. Finney et al (1981) reported
that using falling number or visual reports of sprouting greater than 30% does

not accurately reflect alpha-amylase activity.

At low levels of soft wheat sprouting, Finney et al (1981) reported an
increase in Japanese-type sponge cake volume. Greater than 0.35 dextrinizing
units per gram, however, resulted in extreme volume losses. The authors

proposed that no adverse effects on sponge cake quality could be assured if the

77

amount of sprouted wheat were limited to 2.5%, or if the process were modified

to include 28 rather than 40% water.

Lorenz and Valvano (1981) produced high-ratio yellow cake from one,
two, and four day sprouted soft wheat. One day of sprouting caused increased
cake volume, whereas with two and four days, not only did the volume decrease,
but all external and internal cake characteristics were also compromised. These
findings were explained by the authors in light of work by Lorenz and Kulp
(1981), who demonstrated the ability of alpha—amylase to decrease starch
swelling power, and to allow gelatinization at a lower temperature and over a

narrower range.

Thickening power of laboratory-sprouted soft wheat was found to
decrease with greater time of sprouting prior to flour milling. Gels stored at room
temperature displayed syneresis after three days of sprouting and after five days

when stored at 10°C (Lorenz and Kulp 1981 ).

Finney et al (1980), using field-sprouted soft wheat and composites Of
sound and sprouted wheat, examined the product quality of nine International
breads including: fermented, unfermented, whole grain, and white flour products.
Chapatti made from either a moderately or highly sprouted whole grain flour
composite was coarse, dark in color, and unacceptable to a sensory panel. A
fermented Iranian bread, barbari, was noted to have increased sensitivity to
dough water content. Water absorption was lower for sprouted flour dough and
when the optimum was not used, the breads color was very pale and baking
times were increased. Changing proof time from 7 to 11 minutes improved the
crust color. Moroccan sour-dough bread, made from highly sprouted grain was
whiter, its crumb grain was gummy and it contained many air pockets. Several

Indian breads, Egyptian balady, and Moroccan straight-dough whole wheat

72

bread were determined to be acceptable products when made with sprouted soft

wheat.
Dormancy

If merely quiescent, a seed provided with the appropriate environmental
conditions will germinate, while dormant seeds resist germination under such
conditions. When and for how long a kernel is dormant appears to be controlled
by a complex set of factors. Potential relationships examined have included:
spike and seed characteristics, germination inhibitors, germination stimulators,
and environmental conditions (Nielson et al 1984, Reddy et al 1985). Also,
numerous reports have documented white colored genotypes as having greater
tendency to sprout (Gfeller and Svejda 1969). A more complete understanding
of the factors which cause dormancy is paramount to unveiling the mechanisms

of and eventually controlling PHS.

Physical Factors

Huang et al (1983), using scanning electron microscopy and
autoradiography, reported that white wheat cultivars possessed more separation
between the seed coat and tube cells of the inner pericarp, and a looser
integument structure than red wheat cultivars. Also, the white wheat cultivars
imbibed water faster which penetrated the kernel more than in the red wheat

cultivars.

King (1984 a,b), using 49 wheat cultivars, determined that a two-fold
difference in water uptake over the first two h of imbibition was correlated with

whether or not a seed germinated after 30 h. The following were reported to not

73

be associated with varietal differences in water imbibition rates: seed coat color,
protein content, pericarp thickness, thickness of testa or suberized layer.
However, a study by Moss (1973) has implicated grain hardness and thickness
of the lignified sub-testa suberin layer. In addition, smaller seeds have been
reported to imbibe water faster than larger seeds, but this did not correlate with
the germination rate (rate with which kernels germinated after exposure to water)

(King 1984a).

When examining differences in germination rate for seeds within whole
ears, King and Richards (1984) found that awned wheat ears imbibed water
faster than awnless and germinated 10 to 15 h sooner. However, Mares (1983)
who controlled ear wetting and corrected for differences in grain wetting, also
found differences in seed hydration rates. Thus, suggesting that differences in

ear structure might also affect kernel hydration rates.

The path water takes during seed imbibition has been suggested as the
source of variation in rate of water uptake. The greatest resistance to transverse
water movement in wheat has been reported to be the testa (Hinton 1955). In
barley endosperm, water uptake lags behind hydration achieved by the husk,
pericarp and embryo (Reynolds and MacWilliam 1966). Apparently, water first
travels through the husk to the pericarp and is wicked laterally to the embryo.
Briggs and MacDonald (1983) have provided evidence for this pathway in
barley. They submerged the end distal to the embryo in water while either
maintaining high or low humidity at the proximal end. In the high humidity
treatment, germination occurred, while not in the low. Allowing evaporation to
occur in the low humidity environment apparently prevented the wicking of water

to the embryo.

74

Others have suggested that differences in the amount of grain surface
wax might be a factor related to speed of imbibition. However, primarily visual
reports of the degree of waxiness were initially used to base this correlation on
(Pool and Patterson 1958). lsogenic lines that differed in surface waxiness were
reported to show no difference in hydration rate (King and Richards 1984). Work
has indicated that differences may lie in the fact that waxy lines show a 30-fold
greater rate of evaporation than nonwaxy lines. Recently, wax crystals were
reported to be present in the embryo cavity of red wheat varieties stored for 25
years, but were not seen in white cultivars stored an equal time (Evers and
Kratochvil 1993). These authors hypothesized that the wax, from which these
crystals formed, may play a role in modulating the rate of PHS of wheat; and
further, that the waxy material, which contains over 200 hundred components,
may exist in an amorphous condition, in a region of the kernel which would
prevent water or oxygen from entering the embryo. Over time, this waxy
substance may progressively move toward a crystalline state, and thus create
areas of the kernel with less of a water or oxygen barrier, making germination
more likely. To date, the complete mechanism behind varietal differences in

speed of water imbibition by different grain has not been fully elucidated.

Chemical Inhibitors and Promoters

Exogenously applied gibberellic acid (GA) is able to initiate the
germination of dormant grain kernels. Although GA-responsive genes which
result in increased hydrolytic enzyme activity have been identified, none which
are thought to actually initiate germination have been identified (Jacobsen and

Chandler 1987, Morris et al 1991).

75

Suggested inhibitors of germination have included: phenolics, coumarins,
tryptophan and abscisic acid (ABA) (Miyamoto et al 1961, Paulsen and Heyne
1983, Bewley and Black 1985, Walker-Simmons 1987, Morris and Paulsen
1988, Weidner et al 1993). Currently, however, a direct correlation between the

quantity of any of these substances and dormancy has not been established.

Of the substances mentioned above, ABA has received the most
attention. Beyond a direct correlation to dormancy, others have hypothesized
that a specific balance of GA to ABA may be what controls dormancy (King
1989). This appears doubtful in light of work by Karssen and Lacka (1986).
They identified GA-deficient mutants which develop typical kernel dormancy

periods, that are broken by a prolonged storage period or cool temperatures.

Abscisic acid-deficient or ABA-nonresponsive mutants have demonstrated
that without the effects of ABA, dormancy does not occur (Karssen et al 1983,
Karssen and Lacka 1986). When exposed to applied ABA, germination of
embryos from dormant and nondormant seeds has been reported to be
prevented; the nondormant embryos, however, required more ABA (Walker-
Simmons 1987). Interestingly, however, levels of ABA are similar in dormant

and nondormant mature kernels (King 1989).

Heat-stable proteins are reported to be synthesized for a prolonged
period of time in dormant but not in nondormant kernels exposed to an ABA
solution (Ried and Walker-Simmons 1990). They are very hydrophilic and are
hypothesized to protect proteins during times of hydration or repeated drying
and rehydration. Based on this observation, Morris et al (1991) have suggested
that dormancy may be positively controlled. These authors, using CDNA Cloning
and differential screening to examine this phenomenon, have reported

identifying mRNAs from five gene families which are present in greater amounts

76

in imbibing dormant versus nondormant seeds. All of these transcripts
increased during exposure to ABA. Studies have been initiated to map these
ABA-sensititive Clones which appear to be related to dormancy (Rayfuse et al

1993).

Environmental Factors

A correlation between higher temperatures during grain filling and low
dormancy has been reported in several instances (Belderok and Habekotte
1980, Briggle 1980, Hagemann and Ciha 1981). That is, ears harvested and
kept at room temperature lost dormancy faster than those kept outdoors at
cooler temperatures. However, Briggle (1980) and Mares (1984) have reported
that the reverse appears to be true when mature grain becomes Wet; higher
temperatures during wetting resulted in greater dormancy. Similarly, Plett and
Larter (1986) found developing seed, harvested at from the second to the
seventh week post-anthesis, to have a greater loss of dormancy when exposed

to germination conditions at 17°C, compared to 25°C.

In terms of differences in levels of dormancy between cultivars, George
(1967) reported no difference between cultivars when the germination
temperature was 10°C, while at 30°C all had a deep persistent dormancy, and

at 20°C the dormancy period ranged from 20 to 60 days.

Preharvest Sprouting Methodology

Quantifying and Studying Preharvest Sprouting

Rain-damaged grain presents the potential for economic loss to farmers,
millers and processors. Thus, it is paramount to the cereal industry's financial
viability that accurate, fast, inexpensive methods of Classifying sprouting

damage be developed and adopted. For example, an intense evaluation of

77

Australia's 1983/84 harvest, using the Hagberg falling number resulted in the
down-grading of 2 million tonnes of rain-damaged grain at a cost of over $30

million to growers (Keene 1987).

A large variety of measurements have been employed in the evaluation of
preharvest sprouting. All possess positive and negative attributes depending on
the objectives of the measurement, i.e., what is of interest: the potential use of a
grain in a specific end product, the percentage of kernels that have sprouted, or
the degree to which grain components have been modified by sprouting. In
addition, there is generally a trade Off between methodology speed, cost,
technical skill required, ease of use in the field, and accuracy. Grain sprouting
has traditionally been evaluated visually (i.e., the percentage of kernels with a
ruptured seed coat). Although still used by breeders, visual identification of
sprouting is generally used today merely as an early guide to wheat
classification. That is, visual examination is used to determine if objective
measurements are needed to characterize the grain. The advantage of visual
inspection for signs of sprouting is of course its speed and low cost. However, as
fast as this method is, it is equally as inaccurate in predicting grain's alpha-
amylase activity or the effect of sprouting on product quality (Moot and Every
1990). This, in part, is due to changes in enzyme activity over time. That is, after
hydration occurs levels of alpha-amylase increase slowly at first, then very
rapidly. As a result, grain that appears to contain a low percentage of sprouted
kernels may pose no problem or may contain such high enzyme activity as to
prevent its use in products destined for human consumption. Additionally,
sprouted grain easily lose their germ during handling, thus increasing the

chance of inaccurately characterizing the degree of sprouting.

78

Grain test weight (weight per unit volume) generally decreases with
sprouting due to previous loss from the rupture of seed coats and embryo
growth. However, other grain stress such as disease and temperature extremes
can also decrease grain test weight. The degree to which grain test weight is
correlated with sprouting can also be confounded by variables such as
environmental conditions, grain genotype, and handling conditions. The use of
test weight for judging sprouting damage thus appears to be less than reliable

(Orth and Shellenberger 1988).

The Brabender amylograph has long been used to measure the hot-paste
characteristics Of starches and flours and, because such functional properties
are affected by the amount of alpha-amylase present, it is also used to measure
sprouting damage. In the AACC method #22-10, a flour or starch slurry is slowly
heated from 30 to 95°C, and a viscosity curve is recorded with the initial peak
said to represent alpha-amylase activity (AACC 1983). Amylograph pasting
peaks, however, are also related to other variables: inherent pasting ability of
the starch granules, damaged starch content, susceptibility of undamaged
granules to attack by amylase and the protein and gum content (Meredith 1970,
Dengate 1984). Pasting characteristics of isolated starch and of flour treated
with acid to denature alpha-amylase have been used to differentiate between a
flour's inherent pasting ability and its enzyme activity. However, this is a multi-
step, expensive method of determining the degree of sprouting. Furthermore,
amylograph tracings consist of arbitrary units, which collaborative studies have
demonstrated should not be compared from one amylograph to another (Wilson
et al 1974, Mattewson and Pomeranz 1978). Meredith (1970) suggested that the
amylograph can be considered a useful investigative tool, but it is inappropriate

for screening grain for sprout damage or its potential effect on product quality.

79

The penetrometer test is a method designed to predict the baking
potential of flour or meal (Hagberg 1961, Mitchell 1968). Flour and water
slurries are cooked in a boiling water bath with subsequent cooling. The
distance a cone-shaped penetrometer falls through the cold gel is converted into
sprout units. This is considered to predict bread-baking quality better than the
amylograph, because the strength of cold cooked gel is more related to bread

quality than hot-paste viscosity (Meredith and Pomeranz 1985).

Certainly the most widely used predictor of alpha-amylase activity has
been the Hagberg falling number (Hagberg 1960, Hagberg 1961, Perten 1964).
After milling the grain in a falling number mill to a consistent particle size, a
sample plus water are placed in a glass tube. A stirrer is placed in the tube and
the tube is then placed into a boiling water bath. After the slurry is stirred, the
stirrer is then released. The time required, in seconds, for the stirrer to fall
through the gelatinized suspension plus the stirring time is reported as the falling
number value. As alpha-amylase activity increases, falling number decreases.
As with amylograph measurements, falling number values are dependent on
grain genotype, environment, inherent Viscosity of the starch, temperature of
starch gelatinization, quantity of other grain components present, and the
susceptibility of the starch to enzyme attack. Falling number values have
resulted in sprout-susceptible lines having a falling number lower than its alpha-
amylase content would have predicted (McCaig and DePauw 1983). Work by
Moot and Every (1990) found that falling number identified most, but not all,
sprout damaged wheat that would pose quality problems for commercial bakers.
The grain, inaccurately categorized as sound using the falling number method,

produced test breads with a slightly doughy crumb. Also, Ringlund (1983)

20

reported differences in falling number even when alpha-amylase activity was

constant. This was attributed to differences in starch properties.

Treatment of flour with acid, Silver nitrate, or mercuric chloride have been
,used to limit the shortcomings of the falling number method. That is, the
treatments were done to separate a grain's inherent viscosity from the effects of
alpha-amylase (Meredith 1970, Noll 1985). Finney (1985) developed a method
of eliminating the effect of genotype on falling number. A standard curve was
established which related known spiked alpha-amylase activity with falling
numbers of an unknown flour. Although such methods are an improvement over
the standard falling number method, time and level of training certainly prohibit

their use.

Even with its known inherent shortcomings, falling number evaluation of
grain has remained the US. standard method of evaluating Sprouting damage
(AACC 1983). It has established its respect as a suitable method, because of its
speed, simplicity, reliability in the field, and reproducibility (Tipples 1971).

The inherent difficulties with methods previously discussed have, in part,
resulted in the development of direct alpha-amylase assays. Such

measurements have also been desired for the study of the germination process.

Early alpha-amylase methodology measured enzyme activity using starch
as the substrate. Starch hydrolysis was then determined by measuring a
decrease in viscosity, increase in reducing power, or increase in the color of the

slurry from release of a dye from a dye-bound starch.

Of the direct alpha-amylase measurements, the dye liberation techniques
undoubtedly have been most used. These methods are based on the release of

a dye covalently bound to starch. As the starch is enzymatically cleaved, the dye

27

is released into the solution and measured colorimetrically. The major
differences between the dye liberation methods lies in the dyed substrate used,
and whether extraction and the enzyme reaction is performed in one or two

steps.

The method described by Barnes and Blakeney (1974) uses an insoluble
cross linked dyed (Cibachron Blue) starch substrate, in a two-step procedure.
After being extracted, samples are filtered using a special filtration system,
incubated in a heating block along with the substrate for 15 min, filtered again,

and measured colorimetrically.

Another two-step alpha-amylase method was developed by McCleary and
Sheehan (1987). This method employs a soluble, blocked p-nitrophenyl
maltoheptaoside and requires approximately 15 min. The benefit of this method

lies in that the effects of beta-amylase are removed.

AACC method #22-06 based on work by Mathewson and Pomeranz
(1977, 1978), utilizes substrate tablets containing buffer salts and calcium. This
technique requires only three min to perform, because filtration is not required,
and the enzyme extraction and reaction step take place in one container.
However, because the slurry is not filtered, more care must betaan to ensure
that a finely ground sample is used, than is needed in the dye methods

discussed above.

Henry (1989) examined the use of an insoluble cross-linked dye-labeled
starch and a soluble blocked p-nitrophenyl maltoheptaoside in both, a one- and
a two-step method. The one-step methods required approximately two min and
displayed alpha-amylase activity, which correlated with that of the two-step

assay and the falling number method.

22

Gel diffusion techniques combine a starch substrate into an agarose gel.
Wells are cut into the agar, enzyme extract is placed in the well, and the radial
distance the enzyme diffuses is measured after an incubation period of four to
12 h, depending on the temperature used. The diffusion distance is proportional

to the logarithm of alpha-amylase activity.

Several gel assays allow alpha-amylase diffusion to be visualized by
using iodine staining (Mottonen 1970, Gothard 1976). For example, Hejgaard
and Gibbons (1979) included a dyed starch substrate in an agarose gel, and
measured the diffusion zone after washing away the dye that had become
soluble after incubation. A modification of the basic gel diffusion technique
measures alpha-amylase by pressing halved kernels into the gel surface (Hayter

and Allison 1972).

The gel diffusion techniques Offer several advantages. Variations in
alpha-amylase activity from one kernel to another can readily be identified for
less money than the methods previously discussed. Perhaps more importantly,

these methods offer the ability to screen many samples at one time.

Nephelometric determination of alpha-amylase activity relays on the rate
of decrease in turbidity (light scattering) of a solution containing a dextran and
an alpha-amylase extract of the sample being evaluated. Collaborative testing
demonstrated that a nephelometric method exhibited sufficient accuracy and
reliability to be approved as an official AACC method (AACC 1983 Method #22-
07). The Perkin-Elmer Corporation has made an instrument (Grain Amylase
Analyzer) designed for this procedure. Since its conception this assay has
received several modifications including a substrate Change to beta-limit dextran
to avoid measuring beta-amylase activity, a recorder to follow turbidity, and

fungal alpha-amylase as a reference (Kruger et al 1979, Campbell 1980, Kruger

23

and Tipples 1981). Although the nephelometric method requires a good deal of
laboratory skill and is relatively expensive to perform, its apparent advantage
over other methods is its ability to reliably measure alpha-amylase at low levels

(Kruger 1987).

Techniques have also been reported which use fluorescent substrates or
products to either quantify hydrolase activity or to examine location-specific
enzyme activity. Hydrolase activity is of interest, because both lipase and
protease activities increase during germination and these changes in activity
have been suggested to correlate with alpha-amylase activity during germination
(Tavener and Laidman 1972, Pancholy and Lynd 1972, Jenson and Heltved
1982)

One such method relies on lipase hydrolase of a fluorochrome,
fluorescein dibutyrate (FDB) to butyrate and fluorescent fluorescein (Jenson and
Law 1983, Jenson et al 1984). After cut grain halves are dipped into a solution of
FDB, the degree of fluorescence is examined under low power magnification
using a conventional fluorescence microscope or a Carlsburg Seed Quality
Analyzer. Fluorescence can be quantified with a TLC fluorescence scanner. This

technique has been proposed as a technique for quantifying sprouting damage.

Henry and McClean (1986) measured endosperm modification due to
lipase activity using the FDB seed method, and found it to be unable to predict
sprouting levels of individual wheat and barley varieties. This may in part be due
to cell wall modification being a function of enzyme activity and the level of
resistance to hydrolytic breakdown the cell wall exhibits due to thickness and
compactness. For example, a soft endosperm barley mutant (M-737 from

Minerva barley) displays the same level of alpha-amylase and beta-glucanase

24

activity as its parent variety, but exhibits greater cell wall modification during

germination (Aastrup 1983).

Alternative applications suggested for this technique include a method to
follow the malting process and to predict sprouted grain storage longevity
(Jensen and Heltved 1982). Munck (1987) suggested that the FDB seed method
might be exploited as a means of sorting out light sprouted seeds from sound

seeds, using density gradient tables.

Lipase activity as measured in flour samples using spectrofluorimetric
assays of 4-methyumbelliferyl heptanoate (MUH) and FDB hydrolysis are
techniques suggested as rapid (two to five min), sensitive indicators of
germination (Heltved 1984). Flour samples are suspended in a buffer, MUH or
FDB substrates are added and the fluorescence intensity measured. MUH was
reported to hold the following advantages: greater stability in solution and a
better predictor of lipase and alpha-amylase activity. The authors suggested that
F DB would be more applicable to work requiring visual identification of hydrolytic
activity in sprouted grain, because its fluorescence is much easier to see by eye.

Various immunochemical techniques have also been employed in the
study of sprouting. For example, monoclonal antibodies raised against alpha-
amylase II and labeled with fluorescein were used to examine alpha-amylase
transport and cell wall breakdown in germinating barley seeds (Gibbons 1979,
Gibbons 1980, Gibbons 1981). Enzyme linked immunosorbent assay (ELISA)
was developed to follow the synthesis of an inhibitor of barley alpha-amylase ll
during grain filling (Munck et al 1985). Such methods have the advantage of
being highly specific, and thus, appropriate for investigative work. The use of an
ELISA as a tool for screening grain for sprouting damage has yet to be

examined.

25

There is some concern that traditional methods of reporting PHS, that is
alpha-amylase activity measurements and falling number, may not adequately
predict product quality (Kruger 1990). If grain is exposed to water prior to
harvest, other germinative enzymes with the potential to modify product quality
may also be present in Significant amounts. Many of these enzymes have not
been rigorously studied and their potential importance to cereal processing

Should not be underestimated.

Characterizing Resistance to Preharvest Sprouting

One approach to reducing the impact of preharvest sprouting has been to
develop wheat genotypes with improved resistance to sprouting. As a result, a
variety of methods have been developed to quantify the sprouting tolerance of
breeding lines and cultivars. In order to make comparisons within and between

studies, grain maturity, germination, and sprouting resistance must be defined.

Physiological maturity (PM) of grain is generally said to occur when seed
attains maximum dry weight. Identifying a kernel's stage of maturity is difficult
because there is no clear visual distinction between when grain development
has ended and maturitylis reached. However, Hanft and Wyck (1982) have
reported that PM is correlated with the time when glumes loose their green
pigment, and also with the appearance of the pigment strand in the kernel
crease. These authors suggested that the bottom spikelets be used as an

indicator of PM, because they are the last to mature.

Harvest maturity (HM), assessed by subjectively evaluating grain
firmness, is another point sometimes ascribed to grain maturity in sprouting
studies. Countries, such as Australia and Sweden, define HM by grain moisture
content. However, moisture content at maximum grain dry weight is said to range

from 32 to 44% depending on the environment (Hanft and Wyck 1982).

26

The number of days post-anthesis has also been used to report a grain's
stage of maturity (Mitchell et al 1980). This method may be appropriate when
evaluating the potential a material has for sprouting prior to development of
dormancy, and when studying high prematurity alpha-amylase, apart from
sprouting (Mares et al 1987, Gale and Lenton 1987). However, as Derera (1989)
has pointed out, environmental factors can result in Significant differences in the
rate of kernel development after anthesis, thus making it very difficult to use this

method to screen lines based on a given level of maturity.

A method which has received praise for its accurate depiction of maturity
has been eosin ripeness (Derera 1989). .Weilenmann (1976) describes eosin
ripeness to be the point when water, labeled with eosin red dye, ceases to move
into the stems of a harvested ear. The principle behind this technique is that the
cessation of water flow from plant to kernel correlates with the cessation of dry

matter translocation to the kernel from the plant.

Water Uptake, increased respiration, and enzyme level can all be used to
define germination. However, for ease of measurement, termination of
germination is generally used. That is, either when the testa over the embryo is

ruptured or when a shoot or a root can be seen.

Resistance to sprouting has been reported to be 49 to 64%, dependent
on seed dormancy, with the following factors causing 36 to 51% of the variation:
chaff inhibitors, moisture conditions, and experimental error (Strand 1980). Most
studies Of sprouting resistance have focused on the dormancy factor. In such
cases, sprouting resistant genotypes are defined as those with kernels
displaying a reduced rate of germination under conditions favorable for
germination. Degree of sprouting resistance is generally described by comparing

genotypes being studied to varieties with known sprouting tolerance.

27

Having defined maturity, germination, and sprouting resistance, the next
step in a screening program is to establish the conditions that will be used to
favor sprouting and how sprouting will be measured. When studying preharvest
sprouting, several questions must be answered prior to initiating the study. Will
grain be examined in the intact plant, in the ear apart from the plant or as
individual kernels apart from the ear? If the grain is left with the plant will natural
rain or artificial precipitation be used to wet the kernels? lf kernels are removed

from the plant how will they be wet?

Answers to these questions of course are dependent on the aspect of
sprouting under examination. That is, if grain germinability is of interest, then
kernels apart from the plant should be used. However, if the vegetative
structures (e.g., germination inhibitors in the chaff) impact on grain germinability

are in question, then intact plants should be used.

Although grain sprouted under natural precipitation has been used to
examine preharvest sprouting, the Usefulness of such data is certainly in
question due to the many variables not controlled. For example, frequency and
intensity of rain would all need .to be recorded if grain sprouted under natural

conditions were to be used to establish a variety's propensity to sprout.

Grain in the ear has been examined using a variety of rain simulators. For
example, Sorrells et al (1989) used a sprinkler irrigation treatment in the field.
Others have reported variations of spray being applied to spikes held upright in
a Chamber which possesses temperature and humidity control (King and
Richards 1984, Mares 1987). Providing support for the validity of the misting
methods mentioned above, Mares (1983) reported that the ranking of 11 wheat
cultivars’ propensities to germinate was not affected by the wetting methods

used. However, the rate that roots appeared was dependent on the method.

28

Although it has been suggested that misting systems should simulate natural
rainfall in droplet size and velocity, practically speaking, creating conditions

similar to a specific location would be an arduous task (King 1987).

Both subjective and objective measures have been employed to screen
the sprouting tolerance of lines. A sprouting score developed by McMaster and
Derera (1976) ranks lines using values from one (no visible sprouting) to 11
(fully sprouted). Mean scores of each line are determined after a fixed period of
wetting. A similar method described by Mares (1987) determines a line's
relationship between percent sprouted ears and length of treatment. Rate of
sprouting has also been measured by recording the time required for an

arbitrarily chosen percentage of seeds to germinate.

Assessment of sprouting resistance can also be performed using
methodology developed to study germination. That is, a population's germination
capacity, uniformity, and rate are calculated after placing kernels at a constant
temperature, in excess water, either on filter paper or sand. For example,
germination capacity is the proportion of seeds in a population that are able to
germinate while the degree of germination synchrony is known as germination
uniformity. Bewley and Black (1985) have described germination synchrony
numerically as the coefficient of uniformity. The mean rate of germination of a
population, also described by Bewley and Black (1985), is determined by
calculating the coefficient of the rate of germination. In addition, Goodchild and
Walker (1971) suggested that a polynomial regression method of curve fitting,
using total germination and rate of germination, is an appropriate method to use
when studying seed germination plus biochemical and physiological components

ofseeds.

2.9

Alpha-amylase levels and falling number have also been used in the
screening of genotypes for sprouting resistance. These techniques are
especially important because visual sprouting is not always correlated with

alpha-amylase activity levels.

A more extensive objective measure is the sprouting index developed by
Weilenmann (1976, 1987). Grain is harvested after eosin ripeness is established
and falling number is measured before and after a wetting treatment (falling
number dry and falling number wet). These values are then used in a formula
which allows sprouting resistance to be reported on a scale of one to nine (one =
high degree of resistance; nine = no resistance). When a more detailed
description of sprouting resistance is desired, a sprouting index value is

determined at several different times after eosin ripeness.

Prior to beginning a germination study, it has been suggested that seed
viability be established. Gordon (1980) describes a method which includes the
use of several dormancy breaking conditions: cool temperature, dilute potassium
nitrate, and alternate exposure to light and dark. Other dormancy breaking
techniques that have been employed include seed rupture, seed abrasion and
application of GA or ammonia (Taylorson and Hendricks 1977, Cairns and

deVilliers 1987).

Physiology of Germination

Grain Maturation

Seed formation begins following fertilization. One haploid male nuclei
fuses with the egg nucleus, and another with two polar nuclei forming the diploid
zygote (embryo) and the triploid endosperm nucleus, respectively. The process

of mitosis then leads to cell division with the process, for example, in wheat

30

endosperm completed 14 to 20 days after fertilization (Briaty et al 1979). Cell
differentiation, which results in the formation of tissues and organs, begins prior

to completion of cell division.

Seed development is often divided into three stages, each involving
different biochemical events (Dure 1975). Stage I consists of pollination,
fertilization, and zygote formation. Tremendous metabolic activity during this
period is facilitated by the precursors needed for development (saccharides,
fatty acids, amino acids, organic acids, nucleosides, inorganic ions, and water),
being provided by the parent plant. This stage is finished when cell division is

completed and the embryo is fully differentiated.

During Phase II, storage materials (phytin, starch, protein, and fat) are
layed down and the aleurone layer is formed between the endosperm and the
deveIOping seed coat. The aleurone is a layer of densely packed living cells
consisting predominately of protein. Water content, during the deposit of storage

material, is from 50 to 60%.

Stage III begins when the seed moisture level starts to decrease. The
point of lowest moisture level in unweathered grain correlates with seed maturity
and is from 10 to 15%. During this stage, vascular connections between the
seed and parent are severed and water loss may be accelerated through
membrane modification. During this desiccation process polysomes become
single ribosomes and metabolic activity decreases. Low moisture levels result in
the seed entering either a quiescent or dormant state. In both cases, further

development of the embryonic axis ceases.

Water Imbibition

37

Nondormant seeds experience renewed embryonic growth when exposed
to appropriate water, air, temperature, and light conditions. The seeds also must

not be exposed to excess levels of inorganic salts or toxins.

Germination is said to begin with the imbibition of water by quiescent
seeds and ends with the emergence phase, that is, when a portion of the
growing embryo penetrates the seed coat. Initial water uptake is temperature-
dependent. With each 10°C rise in temperature, water uptake is reported to
increase by 5% (Murphy and Nolan 1982). Under constant conditions, Mayer
and Poljakoff-Mayber (1989) determined the optimum temperature for wheat
germination to be from 15 to 31°C, while the minimum and the maximum ranges
were from 3 to 5°C and 30 to 43°C, respectively. The values for barley and rye
were similar to wheat while temperature ranges for maize and rice germination
were slightly higher. As seeds aged, germination temperature ranges

broadened.

On a fresh weight basis, wheat has been shown to require approximately
45% water in order for 50% germination to occur (Lush et al 1981). However,
maximal germination has been shown to occur only when relative humidities are

greater than 97% at 20°C (Owen 1952).

Biochemistry of Germination

Protein-Degrading Enzymes

Proteolytic enzymes catalyze the hydrolytic Cleavage of peptide bonds.
These enzymes can be placed into two large groups based on their ability to
cleave external (peptidases or exopeptidases) or internal (proteinases or

endopepfidases)bonds.

32

Peptidases are distinguished based on the size of the substrate (di- or
tripeptidases) they cleave, or on the identity Of the terminus (amino- or
carboxypeptidases; omega peptidase for those with a blocked terminus) of the
oligo- or polypeptide they attack. These hydrolases are also categorized
according to the size of the residue they release and from the terminus released
from (aminopeptidases, dipeptidyl peptidases, tripeptidyl peptidases,
carboxypeptidases, peptidyl dipeptidases, dipeptidases, and tripeptidases).

Proteinase Biochemistry

Of the two groups of proteolytic enzymes, proteinases are generally the
rate-limiting hydrolases (Barrett 1986a). That is, the degradation of a protein
begins through the action Of a proteinase and then proceeds, unhindered,
rapidly to completion. The description of the four types of proteinases was
initially based on the catalytic mechanism employed (acid, metal, serine, and
thiol proteinases). However, these names have evolved to reflect the increased
details known about the catalytic sites of these enzymes (aspartic, metallo-,

serine, and cysteine proteinases).

Aspartic Proteinases

Aspartic proteinases (EC 3.4.23) contain an aspartic acid residue in their
active site and in general possess an acidic pH Optima (pH 3.5 to 5.5). Found
only in eukaryotes, aspartic proteinases, have been reported to have molecular
weight range (Mr) values ranging from 30,000 to 45,000. In general they have 3
pl below pH 5.1, however, forms have been reported with pl values up to 6.5

(Barrett 1986a).

Aspartic proteinases seem to act through a general acid-base

mechanism. That is, water attacks the carbonyl of the peptide bond to be

33

cleaved, and the carboxylates in the active site assist with the proton transfer
(Rich et al 1984). These enzymes, in general, prefer to act on peptides bonds
existing between bulky hydrophobic amino acid residues. However, beyond this
general trend in selectivity, this group of proteinases contains those which will
cleave only one specific bond in a given substrate, while others show little

selectivity (Barrett 1986a).

Several aspartic proteinases have been reported to be synthesized as
zymogens which are converted to their active form by hydrolytic cleavage of an
N-terminal extension. For example, pepsin, an enzyme secreted as the zymogen
pepsinogen into the stomach of mammals is responsible for protein digestion.
The increased activity of this enzyme on certain substrates, at as low a pH as
two, is thought to occur not only because the pK of its two catalytic carboxyl
groups is in this region but also as a result of pH stimulated substrate unfolding.
In comparison the pH optima for renin's ability to cleave angiotensinogen in

blood is near neutral depending on the species (Barrett 1986a).

Known inhibitors of aspartic proteinases include diazoacetyl amino acid
esters in the presence of cupric ion (DAN) and 1,2-epoxy-3-(p-nitrophenoxy)-
propane (Stauffer 1987). These inhibitors inactivate aspartic proteinases by
forming an ester with a carboxylate group. An irreversible tight binding inhibitor
of aspartic proteinases is pepstatin, which is synthesized by various strains of
Streptomyces. Enzymes with a low pH optima which are inhibited by DAN and
pepstatin can be fairly comfortably labeled an aspartic proteinase.

Doi et al (1980) isolated an aspartic proteinase from resting rice (Oryza

sativa) with a molecular weight (MW) of 60,000-65,000 and a pH optima Of 2.5-

3.5. Utilizing isoelectric focusing, the enzyme was found to consist of several

34

fractions, all with similar properties. Aspartic proteinases have also been
reported in ungerminated (Wrobel and Jones 1992, Sarkkinen et al 1992, Siuro

et al 1988) and germinating barley (Wrobel and Jones 1992).

Cysteine Proteinases

Animals, plants, eukaryotic microorganisms and bacteria have been
reported to contain cysteine proteinases (EC 3.4.22) (Barrett 1986a). A free
sulfhydryl group and a histidine imidazole group, present in the active site of a
proteinase are the distinguishing characteristics Of cysteine proteinase. Cysteine
proteinase have molecular masses from 20 to 35 kDa, however there are reports
of masses up to 110 kDa. Optimum pH tends to be in the acidic range, but
reports Of cysteine proteinases with optima at 7.5 have also been reported
(Barrett 1986b). In addition to the denaturation of protein substrates, the acidic
pH Optima of cysteine proteinase is thought to be due to the low pKa of the

thiolate-imidazolium ion pair in the active site.

Four superfamilies of proteinases have been identified: the papain
superfamily, the streptoccal superfamily, the calpain superfamily, and the
clostripain superfamily (Barrett 1986b). The papain superfamily is named after
the most studied proteinase of the family, namely, the enzyme papain. This

proteinase is found in the fruit, leaves, and trunk of Carica papaya L.

The activity of cysteine proteinases can be inhibited by thiol-blocking
agents (organomercurial compounds and iodoacetate) and activated by low
molecular weight thiol compounds (dithiothreitol and 2-mercaptoethanol).
Leupeptin and antipain (aldehydes), isolated from Streptomyces strains, and E-
64 (3-Carboxyl-2,3-L-trans-epoxypropyl-leucylamido(4-guanidino) butane, an

epoxide made by AspergiI/us japonicus, have also been reported as cysteine

35

proteinase inhibitors. The former also have an inhibitory effect on serine
proteinase, while the latter binds, irreversibly, to cysteine proteinase only. In
addition a group of polypeptides, known as cystatins, have been identified which
inhibit cysteine proteinase (Rich 1986). Of these naturally occurring inhibitors,

egg-white cystatine is the best understood.

Unlike the animal kingdom, plants primarily use cysteine proteinase rather
than serine proteinase for protein degradation. Of the cereal grains, barley
cysteine proteinases have received the most attention; M of 29,000 to 37,000
have been reported for cysteine proteinase isolated from germinating barley

(Koehler and Ho 1989, Koehler and Ho 1990, Phillips and Wallace 1989).

Metallo-Proteinases

Metallo-proteinases (EC 3.4.24) are found in fungi, bacteria, and higher
organisms (Barrett 1986b). They contain zinc in their active site, are stabilized
by calcium ions, and generally have a pH optimum near seven. As with aspartic
proteases many mammalian metallo-proteinases initially are in the form of a
zymogen, which can be activated either by proteolysis or exposure to

organomercurial compounds.

MetalIo-proteinases frequently hydrolyze a protein between amino acids
with nonpolar side chains (Barrett 1986a). In addition a large hydrophobic
residue (leucine and phenylalanine) is often preferred. Extreme substrate
specificity (e.g. mammalian collagenase) as well as limited selectivity (e.g.

thermolysin from Bacillus thermoproteolyticus) exists in the metallo-proteinase

group.
Identification of metallo-proteinases can be performed using synthetic

substrates such as N-terminus blocking agents like furylacryloyl and

36

phenylazobenzoyl, as well as with various inhibitors. The metal chelator 1,10-
phenanthroline (1,10 PA) has been reported to be an effective inhibitor of
metallO-proteinases (Barrett 1986b). Although also a metallo-proteinase
inhibitor, the use of ethylenediaminetetraacetic acid (EDTA) alone can produce
false results, as serine proteinases are also inhibited by this compound (Powers
and Harper 1986). Other inhibitory compounds considered useful in detecting
metallo-proteinase activity are dithiothreitol (DTT), n-phosphoryl-LeU-Trp,
phosphoramidon, hydroxamic acid derivatives and mercaptoacetyldipeptides

(Barrett 1986a).

A barley proteinase thought to be a metallo-proteinase, with pH optima Of
5.5 and 8.5, and inhibited by EDTA, has been reported (Mikola and Enari 1970).
Using EDTA, 1,10-phenanthroline, and DTT, Wrobel and Jones (1992,1993)
have recently demonstrated persuasive evidence for the presence of five

metallo-proteinases in germinating barley.

Serine Proteinases

Serine proteinases (EC 3.4.21) are undoubtedly the most studied and
widespread of the four classes of proteinases (Barrett 1986b). They have been
identified in prokaryotic and eukaryotic microorganisms, invertebrate and
vertebrate animals, and plants. Two types of serine proteinases have been
identified, namely the trypsin-like (secreted by animal pancreas as a zymogen)
and the subtilisin-like (made by Bacillus sp.), with the former found only in
bacteria. Optimum pH's for these enzymes are generally at seven or higher.
Calcium ions are sometimes required for serine proteinase stabilization, as well

as the activation of their zymogen forms.

37

Serine proteinase substrate specificities are very dissimilar. However,
there are several irreversible inhibitors which Show little nonserine proteinase
inhibition; namely, diisopropyl fluorophosphate (DFP), phenylmethane
sulfonylfluoride (PMSF), and blocked aminoacyl and peptidyl Chloromethanes
(Powers and Harper 1986). Examples of protein serine proteinase inhibitors
includes: soya bean trypsin inhibitor, chymotrypsin inhibitor, and elastase

inhibitor (Ryan and Walker-Simmons 1981).

Barley is the only grain that has been reported to contain serine
proteinases (Wrobel and Jones 1993). The activity of these enzymes was
reported to increase with higher pH conditions and their M was from 47,000 to

91,000.

Proteinases in Wheat

Proteases in wheat have been studied since the early part of this century
(Balls and Hale 1936). During kernel ripening proteolytic activity in three hard
wheat cultivars were reported to decrease 3,4-fold (Bushuk et al 1971 ). Work by
Kruger (1973) found proteolytic activity in two hard wheat varieties to reach a
maximum 16 to 18 days after flowering. At maturity these grains had 50% of their
maximum activity and it was located primarily in the pericarp, seed coat, and

aleurone layer.

McDonald and Chen (1964) found much of the proteolytic activity in wheat
to be extractable using acetate buffer (pH 3.8), water (pH 8.0) or 10% NaCl.
Kaminski and Bushuk (1969), however, reported most proteolytic activity to be in
the alcohol fraction compared to the other Osborne fractions. Using 0.2 M acetic
acid Wang and Grant (1969) were able to extract 75% of total flour proteolytic
activity. Kruger (1973) reported that even after four days of germination,

unextractable proteolytic activity remains in wheat. These differences in reports

38

on proteinase solubility may indicate the following: more than one proteinase is

present and/or bound, as well as, free proteinase is present.

Optimum activity of extractable proteinase from wheat has been reported
to be at pH 3.8 (McDonald and Chen 1964, Wang and Grant 1969). However,
maximum activity for flour was found to be at pH 4.4 (McDonald and Chen 1964).
Mountfield (1936a, 1936b) reported proteinase in germinating wheat to have a
pH optimum of 4.1, at 40°C. All of the proteolytic activity of a dilute acetic acid
extract was reported to be gone at 70°C, while only 50% of the activity was lost

after exposure to 50°C (Wang and Grant 1969).

McDonald and Chen (1964) described the proteinase activity of mature
wheat to be unaffected by trypsin or chymotrypsin inhibitors, while mercury was
reported to have an inhibitory effect. Sulfhydryl blocking agents had a greater
impact on the unextractable than the extractable proteinase. Proteinase activity
in wheat has also been reported to be inhibited by DF P (Belitz and Lynen 1974).
Kawamura and Yonezawa (1982) reported that the addition of pepstatin
completely inhibited the decrease in viscosity of a gluten solution, found after a
24 h incubation. Proteolytic activity in germinating wheat was found to be
increased by exposure to cyanide, while addition of the chloride ion had an

inhibitory effect (Mountfield 1936a, 1936b, 1938).

Attempts to purify the proteinase in wheat appear to have begun with
work by Wang and Grant (1969) and Kaminski and Bushuk (1969). Wang and
Grant (1969) separated an acetic acid wheat extract using gel filtration
chromatography. Two fractions with proteinase activity were identified, one with
a MW of 50,000 and the other was reported to be complexed with glutenin. Four
groups of proteinases, reported to be heterogeneous, were identified using

starch-gel electrophoresis by Kaminski and Bushuk (1969). Skupin and

39

Warchalewski (1971) extracted wheat flour with 10% NaCl, partially purified it
with ammonium sulfate, and separated it into two homogenous, active fractions,
using gel filtration chromatography. The amino acid composition of one fraction
was found to contain disulfide and predominantely unreactive sulfhydryl groups.
Proteinase activity of this fraction was reduced using sulfhydryl blocking agents.
Germinated and quiescent grain extracts were partially purified by Preston
(1978), using affinity chromatography. One peak of proteinase activity was
identified from the quiescent grain, while the germinated wheat extract was
resolved into two heterogeneous peaks. More recently, Lin et al (1993)
produced a crude extract containing proteolytic activity, from a soft wheat, using
ammonium sulfate. One of the two proteolytically active fractions, separated
using gel filtration Chromatography, was found to be related to the change which
occurs in elongational viscosity of cracker sponge during fermentation and was

inhibited by pepstatin.

Intervarietal differences in proteolytic activity have been reported (Bushuk
et al 1971). Proteinases have been reported to not be synthesized de novo;
Mikola and Kolehmainen (1972) suggested that upon tissue. hydration they are
activated. On the contrary, however, others have reported that increased
proteinase activity during germination is at least in part a result of hormonally
induced de novo synthesis (Preston and Kruger 1979, Hammerton and Ho

1986)
Proteinases Effect on Wheat Proteins
After eight days of sprouting, protease activity in hard red spring wheat

endosperm has been reported to increase 17-fold (Hwang and Bushuk 1973).

During this time the amount of insoluble protein decreased and the number of

40

amino groups increased, while no Change was found in the amount of sulfhydryl

and disulfide groups.

Kruger (1980) identified an endoproteolytic system as the hydrolytic factor
involved in storage protein changes during sprouting. Three days of germination

were needed prior to protein Changes, as measured by protein solubility.

Several authors have reported similar trends in Osborne protein
fractionation of sprouted wheat: increased albumin and glutenin fractions;
decreased globulin and residue protein fractions; and no change in the gliadin
fraction (Hwang and Bushuk 1973, Ariyama and Khan 1990). The only difference
found in fractionation studies has been that of Lukow and Bushuk (1984b) who

reported that sprouting resulted in increased gliadin proteins.

Polyacrylamide gel electrophoresis (PAGE) has also been used to try and
further delineate the Changes which occur to wheat proteins during germination.
Two minor albumin bands were reported to appear during germination (Hwang
and Bushuk 1973). Ariyama and Khan (1990) reported only a decrease in
intensity of a few low-mobility albumins with their complete demise occurring
after an extended period of germination. Using sodium dodecyl sulphate
polyacrilamide gel electrophoresis (SDS-PAGE) these investigators also noted
that germinated grain revealed lower intensities for some albumin bands in the

high molecular weight (HMW) range.

Germination has also been reported to result in reduced intensity of a few
HMW globulin bands (Hwang and Bushuk 1973, Ariyama and Khan 1990).
Reduced globulins from sprouted grain showed no quantitative or qualitative

Changes compared to sound grain (Ariyama and Khan 1990).

47

Lukow and Bushuk (1984b) reported an increase in intensity of reduced
gliadins of 118,000, 92,000, and 69,000 daltons while Ariyama and Khan (1990)

found no Changes in reduced or unreduced gliadin bands after sprouting.

No changes were found by Lukow and Bushuk (1984b) in glutenin bands
after grain sprouting. However, Ariyama and Khan (1990) reported various

changes in the intensity of several bands.

Attempting to resolve the apparent disagreement between several of the
studies cited above, Ariyama and Khan (1990) suggested that differences may

have resulted from differences in varieties and/or germination procedures used.

Proteinases Effect on Wheat Product Quality

Many agree that grain would not be fit for food product production, due to
high amylase levels, long before increased levels of proteinase would become a
factor in loss of product quality (Kruger 1990). However, evidence strong
enough to support or reject this opinion is not available. An alternative
hypothesis to the situation is as follows. According to Kulp and Lorenz (1983)
alpha-amylase hydrolyzes grain starch during processing, while proteinases act
both during storage and processing. Therefore it is possible that certain products
may run a greater risk of damage due to proteinase activity. Products which
require little dough handling and are easily penetrated by heat would limit alpha-
amylases effect on product quality. Proteinases, however, having been active
in situ and perhaps during processing, depending on their temperature
tolerance, could become the primary cause of loss of product quality rather than
the alpha-amylase. Examples of products with a potentially greater propensity to
be affected by increased proteinase activity are: flat breads, noodles, and sugar

wafer cookies.

42

Extending the conjecture described above, soft wheat products compared
with hard, may also be more inclined to Show Changes in quality due to
proteinase activity in sprouted grain because of differences in the quality and

quantity of storage proteins.

Edwards et al (1989) reported that defects in the quality of Cantonese
(yellow alkaline) and Korean (white salted) noodles, and Arabic flat breads,
made using five wheat varieties subjected to laboratory wetting, were related to
the flours protease activity. Pan bread, also made in this study, demonstrated
improved quality Characteristics. The authors concluded that the amount and
quality of the protein in the sprouted grain may have influenced the effects seen.
They also reported that protease activity may have played as large a role as
alpha-amylase activity in the loss of product quality. In addition, several authors
have noted that stronger mixing varieties are able to tolerate slight germination
without negatively affecting bread quality (Lukow and Bushuk 1984a, Ariyama
and Khan 1990).

Lorenz et al (1983) examined the functionality of gluten from sprouted
hard wheat varieties, recombined with starch from a control. Gluten from grain
that had undergone low levels of sprouting produced dry extensible dough. More
highly sprouted grain contained gluten which produced a dry inelastic dough.
With greater sprouting, the grain of bread produced from the recombined gluten
and starch became more open and grayer in color. Amino acid analysis revealed
no differences between sprouted and sound grain. The authors concluded that
the changes in gluten functionality, resulting from sprouting, appears to be
related to modification of protein conformation, which occurred primarily in situ,

rather than during the bread making process.

Other Enzymes

43

As seeds germinate, whether in the field or laboratory, many of their
enzymes develop greater levels of activity. Of these, alpha-amylase has been
reported to be responsible for most of the deleterious effects of sprouting on
cereal grain quality. Alpha-amylase de novo synthesis is thought to occur in the
aleurone layer and scutellum, with subsequent release to the endosperm. lts
action on starch results in glucose being released, then converted to sucrose in
the scutellum and transported to the embryo axis to be used for growth (Bewley
and Block 1985, Jacobson 1983, Edelman et al 1959). In addition to alpha-
amylase and proteinases, which were discussed above, the following have also
been implicated as participants in the quality loss resulting from PHS:
arabinoxylanase (Marsh et al 1988), catalases (Kruger 1977),

polyphenoloxidases (Kruger 1976) and lipases (Tavener and Laidman 1972).

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CHAPTER THREE

PROTEINASE ACTIVITY IN SOFT WINTER WHEAT DURING MA TURA TION
AND PREHARVES T SPROUTING

ABSTRACT

Along with alpha-amylase activity, increased proteinase activity has been
linked with the detrimental effects of preharvest sprouting (PHS) on wheat
product quality. However, many questions remain regarding these enzymes.
Those addressed in this study are: what classes of proteinases are present in
maturing soft winter wheat (SWW); is proteinase activity affected by environment
or PHS; and is proteinase activity correlated with alpha-amylase activity or rate
of germination? Five red and four white SWW were grown in a greenhouse
(GHG) and in Dansville, Michigan, in 1992. Samples were harvested at 21, 28,
35, and 42 days post anthesis. Throughout kernel maturation, proteinase activity
was significantly higher in the field-grown grain (FGG) compared to the GHG. At
harvest maturity, proteinase and alpha-amylase activities were positively
correlated (r = 0.669, p < 0.05) in the FGG. Also, germination index was
correlated with proteinase (r = 0.553, p < 0.05) and alpha-amylase activities (r =
0.622, p < 0.05). Utilizing class-specific inhibitors, crude extracts from the
cultivar Augusta demonstrated cysteine and aspartic proteinase activity during
kernel maturation. The cysteine proteolytic activity decreased as kernels

matured, while the aspartic activity increased.

55

56

INTRODUCTION

Sprouting damage, which occurs when rain precedes harvest, is of
concern world wide due to the economic losses which result from decreased
yield, loss of seed viability, and poor flour quality (Tipples et al 1966). Areas of
the world particularly susceptible to preharvest sprouting are Argentina, Brazil,
Chile, Canadian regions of Saskatchewan and Manitoba, Eastern New Zealand,
England, Kenya, Northern Europe, South Africa, and Zimbabwe. For example, in
1987 England realized a $114 Million loss due to winter wheat sprouting (Derera
1990). In the United States, white wheat growing areas including the Pacific
Northwest (Finney et al 1980), New York (Briggle 1980), and Kansas (McCrate
et al 1981), are affected most often. Michigan, primarily a soft white wheat
producer, has also experienced PHS with an estimated 50-60% of its acreage

containing sprouted grain in 1980 (Copeland et al 1980).

The study of proteinases, specifically in soft wheat, is of interest, in part,
because of the potential role they may play in the end-product quality of
sprouted grain. Although increased alpha-amylase activity in sprouted grain has
been thought to be of greater consequence than other enzymes present, a few
studies have linked proteinase activity with a change in product quality (Lorenz
et al 1983, Edwards et al 1989). Also, greater protease activity compared to
alpha-amylase activity in rain-damaged grain has been reported to be carried

over from grain to flour after milling (Edwards et al 1989).

According to Kulp and Lorenz (1983) alpha-amylase performs its
damaging activities during processing, while proteinases act both during storage
and processing. Therefore, it is possible that certain products may run a greater

risk of damage due to proteinase activity. Products which require little dough

57

handling and are easily penetrated by heat would limit alpha-amylases effect on
product quality. Proteinases, however, having been active in situ and perhaps
during processing, depending on their temperature tolerance, could then
become the primary cause of change in product quality rather than alpha-
amylase. Examples of products with a potentially greater propensity to be
affected by increased proteinase activity are: flat breads, noodles, and sugar

wafer cookies (Kulp and Lorenz 1983).

In addition, wheat with lower protein strength has been reported to be less
tolerant to the effects of preharvest sprouting (Ariyama and Khan 1990,
Edwards et al 1989, Lukow and Bushuk 1984). Therefore, soft wheat storage
proteins may be particularly affected by proteinase activity in sprouted grain.
What this means in terms of product quality certainly is not known, as the role of
storage proteins in the plethora of products made from soft wheat is not

completely understood.

The focus of this study was to investigate several questions related to the
hypothesis that proteinase activity, in sprouted soft wheat, results in biochemical
changes which modify endproduct quality. These questions included: (1) what
classes of proteinase are present in maturing soft winter wheat; (2) is proteinase
activity, during kernel maturation, affected by environment; (3) is proteinase
activity, at harvest maturity, correlated with alpha-amylase activity, or degree of
sprouting; and (4) does proteinase activity increase when grain sprouts prior to

harvest?

58
MATERIALS AND METHODS

Materials

Wheat Samples

Samples for this work consisted of five soft red (Cardinal, Mendon,
Hillsdale, Pioneer 2548, Twain) and four soft white (Augusta, Lowell,
Frankenmuth, Geneva) winter wheat varieties (Triticum aestivum). They were
grown during 1991 to 1992, in Dansville, Michigan, and in 1993, in a greenhouse
on the campus of the Michigan State University. The pedigrees of these varieties

are listed in Appendix A.

Chemicals

All chemicals were reagent grade or better and were purchased from

Sigma Chemical Co. (St. Louis, MO).

Methods

Wheat Growing Conditions and Harvest

The experimental design used was two replicates of a completely
randomized block design. One replicate was the field and the other the

greenhouse. Each replicate had two blocks.

Field-Grown Wheat

The field-grown wheat was planted in seven row plots, seven 12 feet long,
with seven inches between rows. The seed was fertilized with 90 lbs N, as urea,

per acre in the spring. Summer temperature and precipitation data collected in

59

the vicinity of Dansville, Michigan by the Michigan Climate Bureau, are reported
in Table I. In each plot, sixty heads were tagged at random throughout the plot,
at anthesis, and 15 heads were harvested from each plot, at 21, 28, 35, and 42
(also called harvest maturity) days post-anthesis (DPA). Upon harvest, 10 heads
from each plot, destined for enzyme work and storage protein analysis were
placed on ice, in polypropylene bags, and within 1 h, stored at -20°C. The
remaining five heads were transported at room temperature to be used later that

day for moisture and germination index measurements.

Greenhouse-Grown Wheat

Seedlings were vernalized for seven weeks, transplanted, and transferred
to a greenhouse. Two blocks containing each genotype were grown. They
consisted of three rows of 10 pots (three plants per pot) per genotype, placed at
random on three benches. Common cultural practices were followed with a 20 h
photoperiod and the temperature ranged from 17 to 25°C. Head tagging and

harvesting were performed as described in the field-grown wheat section.

Sample Preparation

Heads to be used for enzyme analysis were freeze-dried to 4% moisture
or less (freezing time required to reach 4% moisture was first established using
a trial sample from 21, 28, 35 and 42 DPA), threshed in a single-head thresher
and ground using a UDY Cyclone mill with a 0.05 mm screen. Ground samples

were then stored in polypropylene bags at -20°C.

60

Table 1. Temperature and Precipitation in the Vicinity of Dansville,

Michigan, during the Summer of 1992‘

 

 

 

 

Temperature ('F) ° Precipitation (in)

Month Departure 30 Yr

Avg. Max. Avg. Min. From Total Avg-d
Normal“

May 69.9 42.4 -1.0 2.09 2.88

June 73.8 50.6 -3.4 2.07 3.81

July 76.6 55.5 -4.7 6.43 3.91

 

‘ Information provided by the Michigan Climate Bureau.
bAverage maximum and average minimum temperatures.
° NCAA (1982).

d 30 year average.

6 7
Physical Methods

Moisture

Moisture was determined by drying 20 kernels, randomly chosen from the

freshly harvested heads, overnight at 100°C and 30 psi.

Germination Index

The rate of germination was measured using 25 kernels from primary or
secondary florets selected at random from the freshly harvested heads. Kernels
were shaken in 100 ml of 1% sodium hypochlorite for 20 min. They were then
rinsed with 200 ml of water three times and placed, crease side down, on two
layers of Whatman #1 filter paper in a disposable petri dish (100 x 15 mm). Four
ml of water were added to each petri dish prior to placing them into a darkened
cabinet at 20°C and 96% relative humidity (RH). The number of kernels
commencing germination were recorded daily for a period of five days.
Germination was defined as the rupture of the pericarp with at least 1 mm of
growth. A germination index value, for each replication, was calculated by
employing a formula (see Appendix C) reported by Walker-Simmons (1987).
This formula was shortened to five days and values were not divided by the
number of kernels used, because all replications used a consistent number of
kernels. Visible sprouting was evident at 42 DPA in some of the varieties grown
in the field. In spite of this, kernels showing visible signs of sprouting had an
equal chance of being chosen for the germination index and were counted as

being sprouted only after one day in the germination cabinet.

62

Chemical Methods
Protein Content

The protein content of the grain was determined using the AACC micro-
Kjeldahl method #14-13 (AACC 1990).
Alpha-Amylase Activity
Alpha-amylase activity was determined utilizing the AACC method 22-06
(AACC 1990). Reactions were run at 40°C for three min using a dyed Azurine-
cross-linked amylose substrate. After running 10 samples, a sample of Augusta
was analyzed, as a standard, having previously had a mean value plus standard
deviation established. Results for the samples were reported only if the
standard fell within the previously determined mean and standard deviation.
Alpha-amylase activity was reported as dextrinizing units per gram, using a

standard curve established with malt samples spiked with alpha-amylase.

Proteinase Activity

Proteinase activity was measured using azocasein as the substrate. The
basic principles behind this method were first described by Charney and
Tomarelli (1947). One g samples were homogenized with three ml of cold 50
mM acetate buffer (pH 5.4, 10 mM 2-mercaptoethanol) each, using a vortex for
five sec. Enzyme extraction took place by shaking the homogenized mixture at
4°C for 1 h, in 10 ml tubes placed horizontally in a Barnstead Thermolyne
rotatory shaker (1,200 speed). Samples were then centrifuged at 4°C for 20 min

at 25,000 x g, and the supernatants filtered using Whatman GF/A filters. The

63

substrate solution consisted of 1% azocasein in 75 mM acetate buffer (pH 5.4,
10 mM 2-mercaptoethanol), dialyzed against the same buffer overnight at 4°C
and stored in 1 ml portions at -20°C. In 10 sec increments, 150 pl of thawed
substrate was added to the enzyme extract (20 to 150 pl). The amount of
enzyme extract used was established based on the linearity of this assay. After
an incubation period of 3 h at 40°C, the reaction was terminated by adding 0.7
ml of 5% TCA in the same order as the assay initiation. Tubes were then stored
at 4 °C for 20 min, followed by centrifuging at 10,000 x g for 25 min in a micro-
centrifuge. Samples were then read in a spectrophotometer at 366 nm. Blanks
were made by incubating the substrate and sample-extract buffer separately,
and then combining them just prior to terminating the assay. Each time this
assay was run, a standard sample of Augusta was included. Results for the
samples were reported only if the value for the standard fell within the previously
determined mean and standard deviation. Proteinase activity is reported relative
to the value obtained for a sample of the cultivar Augusta germinated for five
days That is, absorbance values were divided by the value found for the
germinated Augusta and the result multiplied by 100. Alpha-amylase and relative
proteinase values are analyzed in the results section using the mean enzyme

activity values for the GHG at each stage of maturity as 100% of activity.

Proteinase Inhibitors

Inhibitor studies were performed using the proteinase activity assay.

Crude proteinase extracts of green-house grown Augusta were incubated with

64

inhibitors at 0°C for 0.5 h in the concentrations listed in Table II. The degree of
inhibition of proteinase activity was calculated by dividing the values from the
assays, which included the inhibitors, by the values from the assays run without
the inhibitor but with the inhibitor solvent (i.e., water, dimethyl sulfoxide - DMSO,

ethanol).

Statistical Analysis
The data were analyzed using the statistical software package Systat

(Systat 1992). Mean separation was performed using the Tukey Test, while
correlation analysis was done by calculating Pearson’s product-moment
correlation coefficients. Tabulated data from this study can be found in Appendix

B.

65

Table 11. Conditions for Proteinase Inhibitor Studies

 

Inhibitor Concentration Solvent

 

Aspartic Proteinases
Pepstatin A 20 pM Ethanol

Cysteine Proteinases

 

 

Iodoacetic acid 10 mM Water
E-64‘ 10 pM Water
N-ethylmaleimide 10 mM Water
CMPS" 5 mM Water
MetalIo-Proteinases
1, 10 Phenanthroline 5 mM DMSO'
EDTA“ 10 mM Water
Serine Proteinase
3, 4 Dichloroisocoumarin 0.1 mM DMSO
PMSF“ 5 mM Ethanol

 

' trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane(L-trans-3-
Carboxyoxiran-2-carbonyl-L-Ieucylagmatine)

° p-chloromercuriphenyl sulfonic acid
° Ethylene diaminetetraacetic acid

" Phenylmethyl sulfonyl fluoride

‘ Dimethyl sulphoxide

66
RESULTS

Inhibitor Studies

Proteinase activity, as affected by class-specific inhibitors, is presented in
Figures 1 to 7. The solvents ethanol and DMSO had an inhibitory effect on total
proteinase activity. Thus the control assays run to compare to the proteinase
activity which existed with the proteinase inhibitors, which were soluble in
ethanol or DMSO, had an equal amount of solvent added to them. Inhibition of
proteinase activity by Pepstatin A was 56% at 21 DPA and increased to 76% by
42 DPA (Fig. 1). Iodoacetic acid inhibited proteinase activity by 36% at 21 DPA
while its effect decreased to 15% by 42 DPA (Fig. 2). Similar to iodoacetic acid,
N-ethylmaleimide and p-chloromercuriphenyl sulfonic acid (CMPS), both showed
a decrease in their effect from 21 to 42 DPA; 49 to 35% and 25 to 20%,
respectively (Figs. 3 and 4). E-64 (3-carboxyl-2, 3-L-trans-epoxypropyl-
leucylamido(4-guanidino) butane) also decreased proteinase activity at 21 and
28 DPA, albeit to a lesser degree than the other cysteine proteinase inhibitors
(Fig. 5). However, 1,10 phenanthroline (1,10 PA), and 2,3 dichloroisocoumarin

(2,3 DIC) had no affect on proteinase activity (Figs. 6, 7).

Enzyme Activity During Maturation

Mean relative proteinase activity of greenhouse-grown soft wheat

cultivars (GHG) versus the field-grown grain (FGG), reported on a dry weight

67

 

 

50 t.‘_ :‘j '

 

" ' ' ' ——No Inhibitor

40 ....... u. Pepstatin A

 

 

 

35-

Inhibitor Effect
D_PA_ % Decrease
21 56

28 71
;, . - £15154; ' ~' 2:: _;;:2,;;;i;._;i»_j, K . ‘j g f 35 75
‘5 - 42 7e

25 f.

20 4:27 " ' . --

 

Relative Proteinase Actlvlty ('l.)

.: ' . " ' l'.. "I 4
10 "" 1‘ '- ~ ": "-v

............
.......
'. .. ‘."."“'"." .........

 

 

 

21 28 35 42
Days Post Anthesis

 

 

 

Fig. 1. Graph of relative proteinase activity of the cultivar Augusta grown in
a greenhouse and harvested at 21, 28, 35, and 42 days post anthesis, with
and without pepstatin A. All assays included ethanol, the inhibitor solvent.
The table on the right of the figure lists the percentage decrease in
proteinase activity which resulted from the incorporation of the inhibitor.

68

 

 

 

 

. —- No inhibitor
, ------- With Iodoacetic Acid

 

 

 

Inhibitor Effect
DPA % Decrease
21 36
28 26
35 18
42 15

 

Relative Protelnase Actlvlty ('l.)

10 L; {11,}: if: 95:52:32.

 

 

 

 

21 28 35 42
Days Post Anthesis

 

 

Fig. 2. Graph of relative proteinase activity of the cultivar Augusta grown in
a greenhouse and harvested at 21, 28, 35, and 42 days post anthesis, with
and without iodoacetic acid. The table on the right of the figure lists the
percentage decrease in proteinase activity which resulted from the
incorporation of the inhibitor.

69

 

 

 

—No Inhibitor ,,- _
....... With N-ethylmaleimide 1‘31?

 

 

 

Inhibitor Effect

DEA $293332
21 49
28 43
35 40
42 35

Relatlve Proteinase Actlvlty (‘/.)

 

 

 

 

21 28 35 42
Days Post Anthesis

 

 

 

Fig. 3. Graph of relative proteinase activity of the cultivar Augusta grown in
greenhouse and harvested at 21, 28, 35, and 42 days post anthesis, with
and without N-ethylmaleimide. The table on the right of the figure lists the
percentage decrease in proteinase activity which resulted from the
incorporation of the inhibitor.

70

 

 

 

——~o
------- With CM 93

 

 

 

lnhf bitor Effect
EA 96 Decrease
21 25
28 21
35 17
42 20

Relatlve Protelnase Actlvlty (‘/.)

 

 

 

 

21 28 35 42
Days Post Anthesis

 

 

 

Fig. 4. Graph of relative proteinase activity of the cultivar Augusta grown in
a greenhouse and harvested at 21, 28, 35, and 42 days post anthesis, with
and without p-chloromercuriphenyl sulfonic acid (CMPS). The table on the
right of the figure lists the percentage decrease in proteinase activity which
resulted from the incorporation of the inhibitor.

77

 

100 .. , . ,. . .

 

   

 

,. —— No Inhibitor
8° ‘é'iiiiieILiF: .1..;;.;:-e.s;:e::;i _ ..:7}T33§fl’f§ -- ~- -. wnn 5.54 3737.322?Eiffii

 

 

 

~. " HT: I 3: :-: .vtf“ . "if. .5 3 .- . - . 1 _
:~ 1: f x: ‘L '

6016 ‘ A

, . lnhibltorEfleCt
% Maw
21 1o
28 8
35 1

42 1

 

Relatlve Proteinase Actlvlty (°/.)
8

20 I. I

10

 

 

 

 

21 28 35 42
Days Post Anthesis

 

 

Fig. 5. Graph of relative proteinase activity of the cultivar Augusta grown in
a greenhouse and harvested at 21, 28, 35, and 42 days post anthesis, with
and without 3-carboxyI-2, 3-L-trans-epoxypropyl-Ieucylamido (4-guanidino)
butane (E-64). The table on the right of the figure lists the percentage
decrease in proteinase activity which resulted from the incorporation of the
inhibitor.

 

72

 

 

 

 

 

 

 

Relatlve Proteinase Actlvlty (7o)

 

 

 

 

21 28 35 42
Days Post Anthesis

 

 

 

Fig. 6. Graph of relative proteinase activity of the cultivar Augusta grown in
a greenhouse and harvested at 21, 28, 35, and 42 days post anthesis, with
and without 1, 10 phenanthroline. All assays included dimethyl sulphoxide,
the inhibitor solvent.

73

 

70 “.3 : 3519155-45..‘:'~"§':;'-'~.;E::"f'fi‘i"-;:':.":}"* 3;'j;t;v:§ ':‘;~‘3'?:?"'f':fif(1‘;j'q'v' ' _'

 

 

 

60 ———~o Inhibitor

 

 

 

j. .. ....... With Dichloroisocoumarin 7155,27

4o f. I i

30 '

 

Relatlve Protelnase Actlvlty (°/.)

20 7;: ' 14., V, ' ‘57- "3:- -'::-:.3;' -' .

   

 

 

 

21 28 35 42
Days Post Anthesis

 

 

 

Fig. 7. Graph of relative proteinase activity of the cultivar Augusta grown in
a greenhouse and harvested at 21, 28, 35, and 42 days post anthesis, with
and without 3,4 dichloroisocoumarin. All assays included dimethyl
sulphoxide, the inhibitor solvent..

74

basis, is displayed in Figure 8. Throughout kernel maturation, proteinase activity

was significantly higher in the FGG compared to the GHG. At 21 DPA,

the FGG had 31% more proteinase activity than the GHG, while at 28, 35, and
42 DPA (i.e., harvest maturity) this difference was 11, 20, and 30%, respectively.

In both the GHG and the FGG, the proteinase activity was 3.3-fold greater at 21

DPA than at 42 DPA. However, the decrease in proteinase activity from 35 to 42

DPA was less in the FGG (14.6%) than the GHG (21.1%).

Figure 9 displays the mean alpha-amylase activity, reported on a dry wet
basis, which was 4-fold higher in the FGG, and 85-fold higher in the GHG, at 21
DPA compared to 42 DPA. In the FGG, compared to the GHG, proteinase
activity was significantly higher at 21 and 28 DPA, the same at 35 DPA and
significantly greater at 42 DPA (Fig. 9). From 35 to 42 DPA, the alpha-amylase
activity in the FGG increased 2.8-fold while the activity in the GHG remained

constant.

Using the moisture data, alpha-amylase and proteinase activity were
calculated on a wet weight basis. Proteinase activity was one-fold higher at 21
DPA compared to the grain at 42 DPA in the FGG, whereas the GHG showed
0.7-fold greater activity. Alpha-amylase activity was found to be two-fold higher

in the FGG and 3.5-fold greater in the GHG at 21 compared to 42 DPA.

75

 

Field
Greenhouse

Relatlve Protelnase Actlvlty ('l.)

 

21 28 35 42
Days Post Anthesis

 

 

 

Fig. 8. Graph of relative proteinase activity in soft winter wheat grown in a
greenhouse and in Dansville, Michigan, in 1992. Values for each location
represent the mean activity of five white and four red cultivars. The relative
proteinase activity of the field and greenhouse grown grain were
significantly different (p < 0.05) at all stages of maturity.

76

 

' m Greenhouse

Dextrlnlzlng Unlts

 

21 28 35 42
Days Post Anthesis

 

 

 

Fig. 9. Graph of alpha-amylase activity in soft winter wheat grown in a
greenhouse, and in Dansville, Michigan, in 1992. Values for each location
represent the mean activity of five white and four red cultivars. Alpha-
amylase activity of the field and greenhouse grown grain were significantly
different (p < 0.05) at all stages of maturity, except at 35 days post
anthesis.

77

Considering all stages of growth and all genotypes, proteinase activity was
positively correlated (r = 0.875, p<0.01) with alpha-amylase activity (Table III).
The protein content of the grain was highly correlated (r=0.930, p<0.01) with the
growing conditions (i.e., higher in the GHG compared to the FGG), however, the
correlation with proteinase and alpha-amylase activities was much lower

“7.0297, p<0,01; r=-0.310, p<0.01, respectively).

Field Environmental Conditions

Table I presents the weather conditions which the field-grown kernels,
used for this study, were exposed to. Temperatures when anthesis occurred and
during the early stages of kernel maturation were less than normal, and on June
22nd, 1992, a frost occurred. Also, during the month of June the area received
46% less precipitation than average. As the kernels moved into the later stages
of maturity, during the month of July, temperatures continued to be lower than
the 30 year average, while precipitation was 65% greater than the 30 year

average.

Field-Weathered Grain

At harvest maturity, the correlation between proteinase and alpha-
amylase activity, in the FGG, was lower (Table IV, r = 0.669) than the

relationship in the grain during maturation, as noted above. Also, germination

78

Table III. Correlation Analysis of Maturing Soft Winter Wheat' Enzyme
Activities with DPA” and Protein Content °

AMYLASEd DPA PROTEIN PROTEINASE,

 

AMYLASE 1.000

DPA -0.683""’ 1.000

PROTEIN -O.310*" 0.144 1.000

PROTEINASE 0.875“ -0.852" -O.297" 1.000

 

‘ Four white and five red varieties were grown in a greenhouse and in Dansville,
Michigan, in 1992.

b Days post anthesis.
° (*) Significant at the p < 0.05; (**) Significant at the p < 0.01.
d Alpha-amylase activity.

° Relative proteinase activity.

79

Table IV. Correlation Analysis of Enzyme Activities with Germination Index
and Protein Content of Mature Soft Winter Wheat Grown in Dansville,
Michigan, in 1992 and Harvested at 42 DPAM

AMYLASE" GERMINATION PROTEIN PROTEINASE“

 

INDEX
AMYLASE 1.000
GERMINATION INDEX 0.622“ 1.000
PROTEIN -0.412 -O.286 1.000
PROTEINASE 0.669" 0.553" -0.287 1.000

 

' (*) Significant at the p < 0.05; (**) Significant at the p < 0.01.
b DPA = Days post anthesis.
° Alpha-amylase activity.

d Relative proteinase activity.

80

index was correlated with proteinase (r = 0.553) and alpha-amylase activities (r
= 0.622). Table V displays alpha-amylase and proteinase in the FGG, at 42
DPA. The alpha-amylase activity found for the cultivar Lowell was significantly
different from all other cultivars. Hillsdale and P2548 had significantly less
proteinase activity than the other seven cultivars, while Augusta and Lowell had
significantly more. Besides varieties, the source of variation found for the alpha-
amylase (p < 0.01) and proteinase (p < 0.01) activities in the FGG, was the

blocks. Appendix B contains the analysis of variance tables for this analysis.
Greenhouse-Grown Grain

At harvest maturity, relative proteinase activity in the nine cultivars grown
in a greenhouse was not significantly different from each other. However, the
cultivar Twain did have significantly greater alpha-amylase activity than the other

cultivars.

DISCUSSION
From the inhibitor studies it appears cysteine and aspartic proteinases

are present during the soft winter wheat maturation process. Cysteine
proteinase activity, as a percentage of total proteinase activity, decreased as the
maturation process proceeded, while aspartic proteinase activity increased.
Previous reports on the classes of proteinase which exist in wheat during
maturation are not available. However, Preston and Kruger (1976) have

reported that as wheat kernels develop, they contain at least two groups of

87

Table V. Alpha-Amylase and Proteinase Activity in Soft Winter Wheat
Cultivars Grown in Dansville, Michigan, in 1992 and Harvested at 42 DPA"b

 

 

Cultivar Alpha-Amylase Relative Proteinase
Activity (DU)° Activity (ti/o)d

we

Augusta 0.38 b 30.69“
Frankenmuth 0.18 b 26.83 "
Geneva 0.29 b 26.72 b
Lowell 0.81a 29.57 "
ge_d

Cardinal 0.11 b ' 27.40b
Hillsdale 0.07 b 25.62 °
Mendon 0.23 b 27.11b
P2548 0.05 b 25.31c
Twain 0.15 b 27.71 ”

 

aValues followed by different letters in a column are significantly different
(p < 0.05).

b DPA = Days post anthesis.
° Dextrinizing units.

d Proteinase activity relative to the activity found in the cultivar Augusta after five
days of germination.

82

proteinases. One group, of acid proteinases, which was more active on
azocasein than hemoglobin, was said to be most active during early
development. Another group which contained neutral proteinases, increased in
activity as maturity approached, and had little activity on azocasein. In terms of
proteinase activity in mature wheat kernels, other authors have reported it to be

partially inhibited by sulfhydryl blocking agents (Skupin and Warchalewski 1971,

McDonald and Chen 1964), while others have found pepstatin A to be effective
(Dunaevskii et al 1990, Kawamura and Yonezawa 1982). These studies, along
with the present work, seem to provide ample support for the presence of both
cysteine and aspartic proteinase in mature grain. Work by McDonald and Chen
(1964) found proteinase to be active both in aqueous solvents and in the flour
left after extraction. They reported the nonextractable proteinase to be more
susceptible to sulfhydryl blocking agents. Kaminski and Bushuk (1969) have
reported most of the proteolytic activity of mature wheat kernels to be in the
alcohol-soluble fraction. The present work, in light of these previous
observations, suggests that during the early stages of maturation, cysteine
proteinases are in a free state (i.e., water soluble). However, as maturation
approaches they become bound to storage proteins, rendering them less
soluble. Thus, saying that proteolytic activity decreases as maturation of a
kernel approaches may not be completely true. A more appropriatestatement
may be that the cysteine proteolytic activity of aqueous extracts of wheat

decreases as a kernel matures, while the aspartic proteolytic activity increases.

83

As found by others and validated by this study, proteinase activities,
reported on a dry weight basis, peak and begin to decline around 20 DPA in
maturing wheat (Cornford and Black 1985, Kruger 1973, Bushuk et al 1971).
The enzyme activity, on a wet weight basis, allows activity at each stage of
maturity to be compared on an equal basis. Although alpha-amylase and
proteinase activities in the present study decreased during maturation, when
considered on a wet weight basis, the decrease was much less than previously

reponed.

The proteinase activity of the FGG at 21 DPA in this study, was close to
that found for the cultivar Augusta, after five days of germination (reported in
chapter four). Evidence that proteinase activity during kernel maturation is
affected by the environment was provided by this study. Research has been
reported which found a correlation between higher protein content and greater
alpha-amylase activity and susceptibility to sprouting (Morris and Paulsen 1985,
Huang and Marston 1980). However, work by Bhatt et al (1981) did not validate
the work mentioned above, nor was a relationship between protein content and
protease activity found. Although all of the GHG had significantly more protein
than their FGG counterparts in the present study, other aspects of the
environment were not controlled. Thus, the effect of a greater protein content
cannot be examined. However, it is fair to say that it appears that if protein
content is correlated with enzyme activity and susceptibility to sprouting as

measured by germination index, the effect must be an interaction with other

84

environmental factors and not nitrogen nutrition alone. That is, the present study
found enzyme activity to be lower in the unweathered, higher protein grain.
Apparently, the environmental factors which influence susceptibility to sprouting

and enzyme activity remain to be fully understood.

Certainly, a question this study raises is: does environment, along with its
effect on proteolytic activity during kernel maturation, affect the synthesis of
wheat storage proteins? Because little is known about the proteinases present in
grain during maturation, such as what metabolic function(s) they perform, the
degree of compartmentalization, and the degree of control by inhibitors present,
it is difficult to postulate about the question posed above. Nevertheless, it may
be speculated that different levels of proteinase in maturing grain can influence
storage protein biosynthesis, as a result of their potential role in peptide
processing during translation. Cereal storage proteins are known to be
translated on ribosomes attached to the endoplasmic reticulum, where
cotranslationally, the signal sequence is cleaved and the polypeptide is released
(Brinegar and Peterson 1982, Yanagata et al 1982). Furthermore, legume
storage protein subunits have been shown to be first cleaved at the N-terminal
signal sequence, and then endoproteolytically cleaved into subunits which are
then linked together with disulfide bonds (Chrispeels et al 1982, Bollini et al
1982). Interestingly, in the current study, proteinase activity in soft wheat was

I

highest during the time wheat storage protein biosynthesis is known to be

completed (i.e., in terms of specific protein patterns on SDS-PAGE being

85

present, but not in terms of band intensity which increased until 35 DPA); more
specifically, around 17 to 21 DPA (Ng et al 1990). Oxidation of these proteins,
that is, formation of inter- and intra-disulfide linkages between them has recently
been reported to occur after storage protein formation is complete and up until
kernel maturation is complete (Gobin et al 1994). Although the proteinase
enzymes involved in the posttranslational cleavage mentioned above have yet
to be identified, the aspartic and/or cysteine proteinases measured in this project
during kernel maturation may be involved. In addition, allelochemical (i.e.,
nonmetabolic) roles for nongerminating proteinase have been suggested. Bell
(1981) proposed a role for plant proteinases similar to that of tannins, that is,
they may participate in defense by degrading parasitic enzymes or toxins.

The summer of 1992, in Dansville, Michigan, was considerably dryer
during the early stages of kernel development while the later period was
exposed to very wet conditions. Such conditions have previously been reported
to be conducive to preharvest sprouting, and its concomitant increased levels of
alpha-amylase activity. In the present study, alpha-amylase activity at harvest
maturity was not as closely correlated to a germination index, as others have
reported (Bhatt et al 1981). Proteinase activity was found to be more closely
correlated with alpha-amylase activity, than a genotype’s propensity to sprout.
By selecting for low alpha-amylase levels, it appears that lower proteolytic
activity should also be expected. Thus reinforcing, as previously suggested, the

value of measuring alpha-amylase, after grain has been exposed to PHS

86

conditions, during cultivar development, in addition to sprouting susceptibility

(Derera 1989).

The need for proper sampling prior to measuring alpha-amylase or
proteinase activity is reinforced by this study. As previously reported for other
grain traits, this study found a micro-environment effect (i.e., differences in
enzyme activity between blocks) to exist for alpha-amylase and proteinase
activity (Finney and Campbell 1994). As a result, the standard deviations for
alpha-amylase were quite large. This resulted in the cultivar Lowell having the
only significantly different level of alpha-amylase activity, even though the
cultivars Augusta, Frankenmuth, Geneva, Mendon, and Twain were found to
have values which would be considered to indicate that sprouting had occurred

(P. L. Finney - personal communication).

Intervarietal differences in proteolytic activity have been reported, as have
increases in proteolytic activity during laboratory germination of grain (Preston et
al 1978, Bushuk et al 1971). However, although many have alluded that
increased proteolytic activity exists in grain due to PHS, limited evidence for this
is available in the literature (Edwards et al 1989). In this study, mean proteinase
activity in the FGG did not increase as maturation approached in response to
PHS conditions, as did the alpha-amylase activity. However, when comparing
the proteinase activity in the grain between 35 and 42 DPA, the activity in the
FGG decreased less than in the GHG. In addition, proteinase activity remained

at a higher level in the FGG at harvest maturity compared to in the GHG. This

87

suggests that grain exposed to PHS conditions will have greater proteinase
activity than unweathered grain. The findings reported by Edwards et al (1989)
appear to be similar to those found in the present study. Three out of five
genotypes exposed to PHS field conditions were reported to have had greater
proteinase activity compared to their controls. However, Bhatt et al (1981)

reported no correlation between field sprouting and proteolytic activity.

A comment regarding the level of alpha-amylase activity in the cultivar
Twain (GHG) at 42 DPA should be made, as it contained significantly more
activity (p < 0.05) than the other eight varieties. The activity in the cultivar Twain
(GHG) increased from 35 DPA to 42 DPA, while it decreased during this time in
the other genotypes. In comparison to the other cultivars, Twain grown in the
field did not display an unusual level of alpha-amylase. This appears to be an
example of a wheat cultivar with high alpha-amylase activity in mature grain,
even when sprouting has not occurred. Such a late maturity high alpha-amylase
(LMHAA) syndrome has previously been reported (Cornford and Black 1985,
Gale et al 1983). Work by Mares and Gale (1990), using the cultivar Spica,
demonstrated that LMHAA is controlled by a single, recessive gene located on
the group six chromosomes and modulated by the environment and the genetic
background. Mares and Mrva (1992) have suggested that greenhouse-ripening
provides better discrimination between high and low LMHAA genotypes,
because some cultivars with LMHAA, depending on the conditions, display low

alpha-amylase in the field.

88

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AACC, 1990. Approved Methods of the A.A.C.C., 9th ed. The American
Association of Cereal Chemists, St. Paul, MN.

Ariyama, T. and Khan, K. 1990. Effect of laboratory sprouting and storage on
physicochemical and breadmaking properties of hard red spring wheat.
Cereal Chem. 67:53.

Bell, AA. 1981. Biochemical mechanisms of disease resistance. Annu. Rev.
Plant Physiol. 32:21.

Bhatt, G.M., Paulsen, G.M., Kulp, K., and Heyne, EC. 1981. Preharvest
sprouting in hard winter wheats: Assessment of methods to detect
genotypic and nitrogen effects and interactions. Cereal Chem. 58:300.

Bollini, R., Van der Wilder, W., and Chrispeels, M.J. 1982. A precursor of the
reserve protein phaseolin is transiently associated with the endoplasmic
reticulum of developing Phaseo/us vu/garis cotyledons. Physiol. Plant.
55:82.

Brinegar, A. C. and Petersen, OM. 1982. Synthesis of oat globulin precursors.
Analogy to legume 11S storage protein synthesis. Plant Physiol. 7021767.

Briggle, L.W. 1980. Preharvest sprouting damage in wheat in the US. Cereal
Res. Comm. 8:1.

Bushuk, W., Hwang, P. and Wrigley, CW. 1971. Proteolytic activity of maturing
wheat grain. Cereal Chem. 48:637.

Charney, J. and Tomarelli, R. M. 1947. A colorimetric method for the
determination of proteolytic activity of duodenal juice. J. Biol. Chem.
171:501.

Chrispeels, M.J., Higgins, T.J., Craig, S. and Spencer, D. 1982. Role of the
endoplasmic reticulum in the synthesis of reserve proteins and the

kinetics of their transport to protein bodies in developing pea cotyledons.
J. Cell Biol. 93: 5.

Copeland, L., Freed, R. and Helsel, Z. 1980. Crop and Soil Science News 6:1.

Cornford, C. A. and Black, M. 1985. Alpha-amylase content of premature
unsprouted wheat grains. J. Cereal Sci. 3:295.

Derera, NP 1990. A perspective of sprouting research. In: Fifth International
Symposium on Pre-Harvest Sprouting in Cereals. K. Ringlund, E. Mosleth
and D.J. Mares (eds). Westview Press, Colorado. pp. 3-14.

Derera, N. F. 1989. Breeding for preharvest sprouting tolerance. In: Preharvest
Field Sprouting in Cereals. N. F. Derera (ed). CRC Press, Boca Raton,
Fl. pp. 111-128.

89

Dunaevskii, Y. E., Sarbakanova, S. T., Belozerskii, M. A. and Zairov, S. Z. 1990.
Simultaneous action of proteases from dormant and germinating wheat
seeds on gliadin. Appl. Biochem. Microbiol. 26:215.

Edwards, R. A., Ross, A. 8., Mares, D. J., Ellison, F. W. and Tomlinson, J. D.
1989. Enzymes from rain-damaged and laboratory germinated wheat. I.
Effects on product quality. J. Cereal Sci. 10:157.

Finney, PL. and Campbell, KG. 1994. Micro-environment effects on plant,
grain, milling, and baking quality. Cereal Foods World 40:575.

Finney, P.L., Morad, MM, Patel, K., Chaudhry, S.M, Ghiasi, K., Ranhotra, G.,
Seitz, L.M. an Sebti, S. 1980. Nine international breads from sound and
highly-field-sprouted pacific northwest and soft white wheat. Bakers
Digest 54:22.

Gale, M. D., Flintham, J. E. and Arthur, E. D. 1983. Alpha-amylase production in
the late stages of grain development - An early sprouting damage risk
period? In: Third International Symposium on Pre-Harvest Sprouting in
Cereals. J. E. Kruger and D. E. LaBerge (eds). Westview Press, Co. pp.
29 -35.

Gobin, P., Kobrehel, K., Bergman, C.J., Ng, P.K.W. and Buchanan, BB. 1994.
Sulfhydryl status of wheat proteins during grain development. In: Gluten
Proteins 1993. Association of Cereal Research, Detmold, Germany, pp.
90-95.

Huang, G. and Marston, E.V. 1980. Alpha-amylase activity and preharvest
sprouting damage in Kansas red and white wheat. J. Agric. Food Chem.
28:509.

Kaminski, E. and Bushuk, W. 1969. Wheat proteases. I. Separation and
detection by starch-gel electrophoresis. Cereal Chem. 46:317.

Kawamura, Y. and Yonezawa, D. 1982. Wheat flour proteases and their action
on gluten proteins in dilute acetic acid. Agric. Biol. Chem. 46:767.

Kruger, J. E. 1973. Changes in the levels of proteolytic enzymes from hard red
spring wheat during growth and maturation. Cereal Chem. 50:122.

Kulp, K. and Lorenz, K. 1983. Changes in baking potential of starch and gluten
wheat flour components caused by preharvest sprouting damage.
Progress in Cereal Chemistry and Technology. J. Holas and J. Kratochvil
(eds). Elsevier, Amsterdam, pp. 847-851.

Lorenz, K., Roewe-Smith, P., Kulp, K. and Bates, L. 1983. Preharvest sprouting
of winter wheat. II. Amino acid composition and functionality of flour and
flour fractions. Cereal Chem. 60: 360.

Lukow, OM. and Bushuk, W. 1984. Influence of germination on wheat quality. I.
Functional (bread making) and biochemical properties. Cereal Chem.
61:336-339.

90

McDonald, CE. and Chen, LL. 1964. Properties of wheat flour proteinases.
Cereal Chem. 41:443.

Mares, D. J. and Gale, M. D. 1990. Control of alpha-amylase synthesis in wheat
grains In: Fifth International Symposium on Pre-Harvest Sprouting in
Cereals. K. Ringlund, E. Mosleth and D. J. Mares (eds). Westview Press,
Co. pp. 183-194.

Mares, D. J. and Mrva, K. 1992. Late maturity alpha-amylase in wheat. In: Sixth
International Symposium on Pre-Harvest Sprouting in Cereals. M. K.
Walker-Simmons and J. L. Ried (eds). American Association of Cereal
Chemists, MN. pp. 178-184.

McCrate, A.J., Nielsen, M.T., Paulsen, GM. and Heyne, E.G. 1981. Preharvest
sprouting and alpha-amylase activity in hard red and hard white winter
wheat cultivars. Cereal Chem. 58:424.

Morris, CF. and Paulsen, GM. 1985. Preharvest sprouting of hard winter wheat
as affected by nitrogen nutrition. Crop Sci. 2521028.

Ng, P.K.W., Slominski, E., Johnson, W.J. and Bushuk, W. 1990. Changes in
wheat endosperm proteins during grain maturation. In: Glutein Proteins
1990. W. Bushuk and R. Tkachuk (eds). Amer. Assoc. Cereal Chem., St.
Paul, MN. pp. 740-754.

NOAA. 1982. National Oceanic and Atmospheric Administration. Monthly norms
of temperature, precipitation, and heating and cooling degree day 1951-
80, Michigan. Climatography of the US No. 81. NCAA, Environmental
Data and Information Service. National Climatic Center, Asheville, NC.

Preston, K. and Kruger, J. 1976. Location and activity of proteolytic enzymes in
developing wheat kernels. Can. J. Plant Sci. 56:217.

Preston, K. R., Dexter, J. E. and Kruger, J. E. 1978. Relationship of
exoproteolytic and endoproteolytic activity to storage protein hydrolysis in
germinating durum and hard red spring wheat. Cereal Chem. 55:877.

Skupin, J. and Warchalewski, J.1971. Isolation and pr0perties of protease A
from wheat grain. J.Sci. Food Agric. 22:11.

Systat. 1992. Systat for Windows, Version 5. Systat, Ivanstown, IL.

Tipples, K.H., Kilborn, RH. and Bushuk, W. 1966. Effect of malt and sprouted
wheat in conventional and mechanical development breadmaking
processes . Cereal Sci. Today 11:9.

Walker-Simmons, K. 1987. ABA levels and sensitivity in developing wheat
embryos of sprouting resistant and susceptible cultivars. Plant Physiol.
84:61.

Yanagata, H., Sugimoto, T., Tanaka, K., and Kasai, Z. 1982. Biosynthesis of
storage proteins in developing rice seedlings. Plant Physiol. 7021094.

CHAPTER FOUR

PROTEINASES IN SOFT WINTER WHEAT DURING GERMINA TION AND
THEIR EFFECT ON STORAGE PROTEINS

ABSTRACT

Characterization of proteinases is needed to understand their role in
kernel physiology, thus opening up possibilities of exploiting them for the benefit
of the food industry. The objectives of this project were to gain a “big-picture”
perspective of proteinases in germinating soft winter wheat (SWW). Two SWW
varieties were germinated for five days. The variety with a greater susceptibility
to sprouting contained more proteinase activity at all stages of germination.
Utilizing class-specific proteinase inhibitors, cysteine proteinase activity
increased, while aspartic proteinase activity decreased until the second day of
germination. These trends then reversed. After one day of germination serine
and metallo-proteinase activity began to increase and peaked after four days.
Ten bands of proteinase activity were visualized using native polyacrylamide gel
electrophoresis with copolymerized gelatin and crude extracts from quiescent
and germinated kernels. Two bands were present in quiescent grain at near
neutral pH, suggesting that they contain serine or metallo-proteinase. The other
eight bands had acidic pH optima and appeared after germination had
commenced, indicating that they contain aspartic or cysteine proteinase.

Glutenin subunits in germinated kernels appeared to be hydrolyzed by

91

92

proteinases earlier than the gliadin proteins. This study demonstrates that

germinating soft wheat contains a complex set of proteinases.

INTRODUCTION

Proteases in wheat have been studied since the early part of this century
(Balls and Hale 1936). However, much about these enzymes still eludes us.
Such as: what classes of proteinases are present during the various stages of a
kernel's life cycle; where are they located in the kernel; what are their substrate
specificities; what are their temperature and pH optima; and where are the

genes located which code for them?

Endogenous wheat enzymes have the potential to create desirable, as
well as deleterious changes in a product, depending on the product’s different
functional property requirements, and thus, different needs for biochemical
components. For example, durum wheat varieties have been selected for low
lipoxygenase activity in order to limit carotenoid pigment oxidation (Hoseney
1994). A potential application in soft wheat, previously identified, lies within
saltine cracker production. Endogenous proteolytic enzymes in flour have been
reported to be related to the desired changes the rheological properties of
cracker doughs undergo during fermentation (Pizzinatto and Hoseney 1980).
Also, increased proteolytic activity which accompanies preharvest sprouting
(PHS) has been linked to changes in product quality (Edwards et al 1989,
Lorenz et al 1983). The examples cited above indicate that proteinases in wheat

play a role in product quality, and thus, warrant further investigation.

Attempts to purify the proteinases in wheat appear to have begun with
work by Wang and Grant (1969), and Kaminski and Bushuk (1969). Wang and

Grant (1969) separated an acetic acid wheat extract using gel filtration

93

chromatography. Two fractions with proteinase activity were identified, one with
a MW of 50,000 and the other was reported to be complexed with glutenin. Four
groups of proteinases, reported to be heterogeneous, were identified using
starch-gel electrophoresis by Kaminski and Bushuk (1969). Skupin and
Warchalewski (1971) extracted wheat flour with 10% NaCl, partially purified it
with ammonium sulfate, and separated it into two homogenous active fractions
using gel filtration chromatography. Proteinase activity of one of these was
reduced using sulfhydryl blocking agents. Germinated and quiescent grain
extracts were partially purified by Preston et al (1978) using affinity
chromatography. One peak of proteinase activity was identified from the
quiescent grain, while the germinated wheat extract was resolved into two
heterogeneous peaks. More recently, Lin et al (1993) separated a crude extract
from soft wheat using gel filtration chromatography. They reported two fractions

with proteolytic activity, one of which was inhibited by pepstatin.

The objectives of this study were to gain a "big-picture" perspective of the
proteinases in quiescent and germinating soft wheat, and to characterize their

effects on soft wheat storage proteins.

MATERIAL AND METHODS

Materials

Wheat Samples

Augusta (a soft white winter wheat) and Hillsdale (a soft red winter wheat)
provided by the Department of Crop and Soil Sciences, Michigan State

University, were used for this study.

94

Chemicals

All chemicals were of reagent grade or higher and were purchased from

Sigma Chemical Co. (St. Louis, MO).

Methods

Wheat Growing Conditions and Harvest

Seedlings were vernalized for seven weeks, transplanted, and transferred
to a greenhouse. Two replications of each genotype were grown, consisting of
three rows of 10 pots (three plants per pot) per replication, placed at random on
three benches. Common cultural practices were followed, with a 20 h
photoperiod and the temperature ranged from 17 to 25°C. Heads were tagged at
anthesis, harvested 42 days later, placed in paper bags, and stored at room

temperature.

Sample Preparation

Controlled Germination

Grain samples of greenhouse-grown (GHG) Augusta and Hillsdale (70 g
each) were surface sterilized according to Hoy et al (1981) with some
modifications. Kernels (10 g per container) were shaken in 200 ml of 0.1% (w/v)
silver nitrate for 20 min, then washed three times with 200 ml of sterile 0.5 M
NaCl, followed by the same routine using sterile water. The kernels were then
soaked for 24 h under agitation, using 100 ml of sterile water per container. The
sterile water contained 10,000 U penicillin, 10 mg streptomycin, and 25 pg
amphotericin B per ml. After draining the antibiotic/antimycotic soak water, each
10 9 lot of kernels was placed on two layers of blotting paper in a flat bottomed

container and germinated in the dark at 20°C and 96% RH. At the end of each

95

germination period, samples were freeze-dried, root and shoot growth was
manually removed and the kernels were ground in a UDY Cyclone mill (0.05 mm

screen).

Treatments for this study consisted of the two controls, Augusta and
Hillsdale (A—CL, H-CL), kernels soaked 24 h (A-24h, H-24h), and kernels
exposed to conditions condusive to germination for 1 (A-ID, H-1 D), 2 (A-2D, H-
ZD), 3 (A-3D, H-3D), 4 (A-4D, H-4D), and 5 days (A-5D, H-5D). It should be
understood that, in the text, when referring to grain exposed to conditions
conducive to germination, not all kernels at a given time were germinating.
However, for ease of discussion, the samples will be referred to as having been

germinating for a given number of days.
Milling

Heads to be used for enzyme and storage protein work were freeze-dried
to 4% moisture, threshed in a single-head thresher, and ground using a UDY
Cyclone mill with a 0.05 mm screen. Ground samples were then stored in

polypropylene bags at -20°C.

Germination Index

A germination index was measured for the cultivars Augusta and Hillsdale
following the method decribed in chapter three of this dissertation. This index

was measured one month after the grain was harvested at 42 DPA.

Proteinase Activity

Proteinase activity was measured using the method described in chapter
three of this dissertation. All values are expressed relative to the maximum value

obtained in this study, which was for A-5D.

96

Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-
PAGE) of Glutenins

A method by Ng and Bushuk (1987) was used to examine the effects of

sprouting on glutenin subunits and is described in Appendix C.

Acid Polyacrylamide Gel Electrophoresis (PAGE) of Gliadins

Gliadins were examined using the first dimensional procedure of the two-
dimensional PAGE method described by Lafiandra and Kasarda (1985). The
extraction component of this method was not used; instead samples were
extracted following the method by (Ng and Bushuk 1990). The modification of
this method consisted of the use of 50% sucrose and 3% w/v methyl green dye
in the extraction solution. A description of the acid-PAGE procedure can be

found in Appendix C.

Proteinase Characterization

The wheat samples used to characterize the proteinase in soft winter

wheat were Augusta and Hillsdale, germinated from 1 to 5 days.

Enzyme Extraction

Samples were homogenized with 50 mM acetate buffer (pH 5.4, 2 mM
cysteine) in a 1:2 ratio (g/ml), using a polytron for 2 min. Slurries were then
extracted by shaking for 1 h, centrifuging at 5,000 x g for 20 min, and dialyzing
(MWCO 12-14,000) overnight, against 10 volumes of 5 mM acetate buffer (pH
5.4). The crude extract was then pipetted into 100 ttl lots and stored at -20°C. All

procedures mentioned above were carried out at 4°C.
Native PAGE with Copolymerized Substrate

Proteinases were separated and visualized using PAGE with copolymerized
gelatin (0.1% in water) or glutenin (0.1% in 0.05 M acetic acid), according to

Wrobel and Jones (1992). Modifications to this method included the use of 25 ttl

97

of sample extract per well, gel incubation at pH 3.8 (11 h), 5.4 (18 h), and 6.5
(18 h), and the use of glutenin as a copolymerized substrate. This method can
be found in its entirety in Appendix C. Only crude extract from the Augusta

samples were run on the gels with copolymerized glutenin.

Glutenin Preparation

The preparation of glutenin for use as a substrate in the proteinase gels
involved several steps. First, crude gluten was prepared according to AACC
method 38-10 (AACC 1990) from 10 g of Augusta meal which had been grown in
a greenhouse and harvested at 42 DPA. Second, the crude gluten was stirred
with 50 ml of 0.05 M acetic acid for 1 h at 4°C. The solubilized glutenin was then
separated from the insoluble material by centrifuging the mixture at 5,000 X g for
10 min at 4°C. The supernatant was then dialyzed (MWCO 12,000 - 13,000)

against distilled water, overnight at 4°C, and freeze-dried.

Proteinase Inhibitor Studies

Inhibitor studies were performed according to the method described in
chapter three of this dissertation. The crude extract used was from the

ungerminated and germinated Augusta grain described above.

Statistical Analysis

The data were analyzed using the statistical software package Systat

(Systat 1992).

98
RESULTS

Germination Index

The germination index calculated for the cultivar Augusta was 130.
However, the germination index found for the cultivar Hillsdale (40) was less

than one third of that found for Augusta.

Proteinase Activity

Proteinase activity is reported in Figure 10. All values are expressed
relative to that which was obtained for A-5D, that is, as a percentage of the
highest absorbance found during this study. After the 24 h soak through five
days of germination, the Augusta samples displayed significantly (p < 0.05)
greater proteinase activity than the Hillsdale samples. From one to five days of
germination, activity in Augusta increased at a faster rate than in Hillsdale.
Proteinase activity was 6.7-fold greater in the A-SD compared to A-CL, while H-
5D had 3.5-fold greater activity than the H-CL. After five days of germination,

Augusta was found to have 1.7-fold greater activity than Hillsdale.

Visualization of Proteinase Activity

The proteinase activity bands (zones of hydrolysis) of the gels with
copolymerized gelatin were numbered in the following manner: the band which
had the fastest mobility was labelled B-10 (A-2D incubated at pH 3.8). All
others, appearing in ascending order toward the top of the gels, were labeled B-

9 through B-1 (Fig. 11).

99

 

   

Relatlve Protelnase Actlvlty (7.)

Augusta

 

 

 

Fig. 10. Graph of the relative proteinase activity of the cultivars Augusta
and Hillsdale grown in a greenhouse and exposed to conditions condusive
to germination in a laboratory. Control is the ungerminated grain, 24 h is
the grain after soaking for 24 h, and 1 D through 5 D is the grain
germinated from one to five days. All Augusta values are significantly
greater (p < 0.05) than the Hillsdale values, except for the controls.

700

Fig. 11. PAGE with copolymerized gelatin of soft wheat crude extracts
prepared from grain at different germination stages. A, cultivar Augusta; H,
cultivar Hillsdale; lane 1, ungerminated; lane 2, 24 h soak; lanes 3, 4, 5, 6,
and 7, 1 through 5 days of germination, respectively; pH 3.8 (0.1 M acetate
buffer, 11 h), pH 5.4 (0.1 M acetate buffer, 15 h), and pH 6.5 (0.1 M sodium
phosphate buffer, 15 h), pH of gel incubation buffers; B1 to 811, enzyme
band code. Buffers all contained 2 mM cysteine. Twenty-five microliters of
extract loaded per gel.

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Two bands (B-1 and B-2) of proteolytic activity in both quiescent grains
(A-CL and H-CL) could be seen when the gels were incubated at pH 5.4 and 6.5.

Increased activity of these bands became evident as germination progressed.

The Augusta gel, incubated at pH 3.8, first showed signs of activity after
one day of germination (A-1D). After two days of germination (A-2D), two
additional bands (B-3 and B-10) appeared which disappeared at three days of
germination (A-3D). From day two to day three of germination, an increase in
the proteinase activity of the five bands present (B-4, B-5, B-6, B-7 and B-8)
was noted. This level of activity for all five bands appeared to remain constant
throughout the duration of the germination period. Four bands (B-4, B-5, B-6 and
B-8) were noted in the Augusta gel incubated at pH 5.4 after three days of

germination. There seemed to be no change in these bands after four or five

days of germination.

Proteinase activity first appeared in the Hillsdale gel incubated at pH 3.8
after three days of germination. These two bands of activity (B-5 and B-8)
remained during the fourth and fifth days of germination. At pH 5.4, the Hillsdale
gel showed only a slight amount of activity, in the faster mobility area, at five
days of germination. A small amount of hydrolysis occurred here, but it is not

visible in Figure 11.

The gels containing copolymerized glutenin photographed poorly due to
low activity, and thus, are also included in this document. However, three faint
activity bands were evident on the gel incubated at pH 3.8 after three days of
germination. These bands were more diffuse than the bands on the gels with
copolymerized gelatin. One band had faster mobility than any of the bands on
the COpOIymerized gelatin gels. The other two bands appeared in the same

regions as BS and B6. Proteinase activity was evident on the gel incubated at

703

pH 5.4, but only after four days of germination. No proteinase activity was seen

on the gel incubated at pH 6.5.

In summary proteinases detected in the quiescent grain displayed
increased activity with germination, had slow rates of mobility on a native PAGE
gel, and appeared to have pH optima close to neutral. Proteinases with activity
bands that appeared after germination had faster mobilities and acidic pH

optima.

Inhibitor Studies

Figures 12 to 18 contain graphs displaying the effect of various inhibitors
on the proteinase activity of Augusta crude extract from quiescent grain, grain
soaked for 24 h, and grain germinated for one to five days. Crude extract
proteinase activity when incubated with pepstatin A was inhibited 72% in the
control. The level of inhibition decreased to a low of 38% by the second day of
germination, then increased to 70% after four days of germination, and fell to
52% after five days of germination. Iodoacetic acid, 3-carboxyl-2, 3-L-trans-
epoxypropyl-leucylamido(4-guanidino) butane (E64) and N-ethylmaleimide all
demonstrated increased inhibitory effectiveness from the ungerminated stage
(14, 1, and 30%, respectively) until day two of germination (61, 45, and 50%,
respectively). After the second day of germination, their percentage of inhibition
continued to fall until the final day of germination. After five days of germination,
however, their inhibitory ability was greater than the degree of inhibition seen at
quiescent stage. Similarly, p-chloromercuriphenyl sulfonic acid (CMPS)

increased in its ability to inhibit proteinase activity beginning during the 24 h

704

 

Relatlve Proteinase Actlvlty ('/.)

 

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SD

Inhibitor Effect
Time % Decrease

Control
24h
1D
20
3D
4D
50

72
69
53
38
55
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52

 

 

Fig. 12. Graph of relative proteinase activity of the cultivar Augusta, grown
in a greenhouse, with and without pepstatin A. All assays included ethanol,
the inhibitor solvent. Control, ungerminated grain; 24 h, grain soaked for 24
h; 1 D, 2 D, 3 D, 4 D, and 5 D, grain germinated for one to five days. The
table on the right of the figure lists the percentage decrease in proteinase
activity which resulted from the incorporation of the inhibitor.

705

 

 

100 ‘. ' . . .,

   
  

   
 

 

 

 

 

80 ------- Nth Iodoacetic Acid if;.-?f'::}:t:f-z*:~;)3}

 

70 -iii‘5:=.-;ff§::;ft'

Inhlbltor Effect
TILE; % Decrease
Control 14
24 h 23
1 0 40
‘ 2 D 51
40 "“ 22391:; T 753:; Psi-iii:25;}liiri 3 D 51

_ f kl? 4 0 45
30 ~é-g~=.:.ii?*ii<‘? .9‘i§*ii;i.i’i:~ Tic-g: 5 D 46

60 "

 

;. :‘ ;.' .‘.' . . ‘ '." ' v C‘- ' Z , . r’.' . .'.'.'
r . . , ,1 .> .7 . . ,. 1.. .‘ .. ‘. .' .' n',
‘.V‘; .' . . ._.> ' ,,' .1 .I . . ., .'.’ . .. I" '. .‘ . . ', " ',

,, .' " ' ‘ , . . , . ‘.'.‘ ‘ . - _’ ‘. . ,,' , ‘ ‘ , I \ '~ ‘ ,

Relatlve Proteinase Activity ('l.)

20

 

10 . . I
O .5 I . T ' .
Control 24h ID 20 3D 40 SD

 

 

 

 

 

 

Fig. 13. Line graph of relative proteinase activity of the cultivar Augusta,
grown in a greenhouse, with and without iodoacetic acid. Control,
ungerminated grain; 24 h, grain soaked for 24 h; 1 D, 2 D, 3 D, 4 D, and 5 D,
grain germinated for one to five days. The table on the right of the figure
lists the percentage decrease in proteinase activity which resulted from the
incorporation of the inhibitor.

706

 

 

Relatlve Protelnase Actlvlty (7.)

 

so ,5; ‘ "

 

so “3:, Q‘

 

 

 

 

0 29252.“".’"”

No hhibitor E 7*." E H
....... with E-64

100 :5." ' " r :f.'- . j ; '7:::.~"5:,';4_; ~

so I

40 v , - .-
' " t

. v .V,‘ ‘ ' V V ' . . .~ ,E. i v V v ‘1 i‘v ‘ . . . . V ‘ ,~ . ,_ ‘ , ' . .. . . . I. . , .' i
l
. . . v .' . ‘ . ‘ r '- . I 7 . ' . ,' ‘ E ’ . . V E 7, '1 '- . . . ‘ . ,
. . '~ .‘ '. :.Z' .l‘ " 13"» .> - '.... v ' . .
l. .A. " .V, E, -' .'. ‘7 ‘ . > ~. » ‘.I . 7. ‘ ‘li ‘. V E >’. . i . A .' ‘ '1 E '. '.‘ ‘
. . . . . , \ " v ’ . ,‘U‘f' - .': 7 ~ .’ ' ‘- E l - ‘7 V V I '

1° ._

  
    

.- Control
24 h

1 D

2 D

3 D

4 D

5 D

 

 

Control

 

24H

20

Time

 

30

o‘— -——

40 SD

. InhibitorEffect
'“"'>**”."’.E~y-§’.' Time % Decrease

1
6

24
45
1 7
30
36

 

Fig. 14. Graph of relative proteinase activity of the cultivar Augusta, grown

in a greenhouse, with and without 3-carboxyI-2, 3-L-trans-epoxy propyl-
Ieucylamido (4-guanidino) butane (E-64). Control, ungerminated grain; 24 h,
grain soaked for 24 h; 1 D, 2 D, 3 D, 4 D, and 5 D, grain germinated for one
to five days. The table on the right of the figure lists the percentage
decrease in proteinase activity which resulted from the incorporation of the

inhibitor.

 

707

 

 

1m i" ‘1 '.-;~'1:‘.-:-:?‘3,-.-;11 . . . “33.7. :1 313' 2 1 775 1» '

Nolnhihitor j .
5552'5'2155151151'79 ------- h N- th I l ' '
8° ‘E..II*”:3*°L . 1-. wt. _e. in“ ”W? i' .1:-..-~i'f:. 7 - '- ' “ ‘

 
  
 

 

 

 

 

 

 

70 .., ' .2

I nthItor Effect
In: °_II; Decrease
Control 30
24 h 40
1 D 41
2 D 50
3 D 44
4 D 25
5 D 41

Relative Proteinase Activity (7.)

30 .
'1. ,;~: v
20 «ii-":1? .-7f?3“fj'ift'igii‘255 s1] ' " ” ‘ " " " ‘
“n 5.15 ‘5 ~ 1’ V, ' V H

10 4:11.:1 'i' 3,3' f 0: ‘~: ‘1-“.-',:'~':.-;?.’.~,' . ' ’ '2 ' ‘
-:-.:-.

   

 

 

 

o ' ‘
Control 24h 10 20 30 40 50

Tim e

 

 

 

Fig. 15. Graph of relative proteinase activity of the cultivar Augusta, grown
in a greenhouse, with and without N-ethylmaleimide. Control, ungerminated
grain; 24 h, grain soaked for 24 h; 1 D, 2 D, 3 D, 4 D, and 5 D, grain
germinated for one to five days. The table on the right of the figure lists the
percentage decrease in proteinase activity which resulted from the
incorporation of the inhibitor.

708

 

 

— No Inhib'tor ['2

3 ....... With CNIPS 1..

 

 

E?

S

g Inhibitor Effect
2 En: 22mm
3 Control 20

E 24 h 30

g 1 D 38

i 2 D 15

g 3 o 14

E 4 o 11

g 5 o 14

 

 

Control 24h ID 20 3D 4D SD

Time

 

 

 

Fig. 16. Graph of relative proteinase activity of the cultivar Augusta, grown
in a greenhouse, with and without p-chloromercuriphenyl sulfonic acid
(CMPS). Control, ungerminated grain; 24 h, grain soaked for 24 h; 1 D, 2 D,
3 D, 4 D, and 5 D, grain germinated for one to five days. The table on the
right of the figure lists the percentage decrease in proteinase activity which
resulted from the incorporation of the inhibitor.

709

 

 

 
    
 

 

------- With 1. lo phenanthroline i32.122%:-;2-¥-‘-"-f:'i§:—J--I¥::15?" 295‘2i291222.255:"‘Piiz’f’:

 

 

 

 

 

60 . ;

Inhibitor Effect

Tl_rm_ % Ecrgase
33? ‘.:.:;:”5:3.{'?. .3973?“ .f:lia-iilfi'.".:£. 1'71 Control 1
4O -éi5?3?ffi5?§iééi275‘??? .:-;§T:;i*5}¥it'l*7i‘s - 4:95" . ~-ii::-;L-i2{.:4i 24 h 1
' -~11]?1.2111523;f7f;~s;'>}1;¥:‘2.213313: 2:33.41" ,2; :i' 1 o 7
igi » 1 V 1 2 D 13
3 o 13
4 o 26
5 D 10

30

Relatlve Proteinase Activity (7.)

zo

10

 

 

 

Control 24h 1D 2 D 3 D 4 D 5 D

 

Time

 

 

 

Fig. 17. Graph of relative proteinase activity of the cultivar Augusta, grown
in a greenhouse, with and without 1, 10 phenanthroline. All assays included
dimethyl sulphoxide, the inhibitor solvent. Control, ungerminated grain; 24
h, grain soaked for 24 h; 1 D, 2 D, 3 D, 4 D, and 5 D, grain germinated for
one to five days. The table on the right of the figure lists the percentage
decrease in proteinase activity which resulted from the incorporation of the
inhibitor.

770

 

 

80

   

 

70 No inhibitor E ’_
_f 2.21.5.1); . . . . .. . . Wth 1, 10 phenanthroline {1 f .' . 1

 

 

 

 

60 : -

Inhibitor Effect
M '5 Ecrgase
, . . ., _ .2 ._ , ....,-?- ;:'j:g~ ; . CONVOI 1
* ' 1o 7
2 D 13
3 D 13
4 D 26
5 D 10

Relative Proteinase Actlvity ('l.)
8

1o ‘5?[;*.2_;yiialI g * , * ' "

 

 

0 51:51:17," " f _ ’- 3-.:,=E'111§_I13fi ‘ ‘ 7 l. I, . 1 ,
Control 24h 10 20 3D 4D 5D

 

Time

 

 

 

Fig. 18. Graph of relative proteinase activity of the cultivar Augusta, grown
in a greenhouse, with and without 3,4 dichloroisocoumarin (3,4 DIC). All
assays included dimethyl sulphoxide, the inhibitor solvent. Control,
ungerminated grain; 24 h, grain soaked for 24 h; 1 D, 2 D, 3 D, 4 D, and 5 D,
grain germinated for one to five days. The table on the right of the figure
lists the percentage decrease in proteinase activity which resulted from the
incorporation of the inhibitor.

777

soak, but its maximum inhibitory effect was found after one day of germination
(38%). Both, 1, 10 phenanthroline (1,10 PA) and 3, 4 dichloroisocoumarin (3,4
DIC) began to have an inhibitory effect some time between the 24 h soak and
the first day of germination. Maximum inhibition for 1,10 PA and 3,4 DIC, that is,

26% and 14%, respectively, was found to occur after four days of germination.

Proteinase Effect on Storage Proteins

Figure 19 displays the SDS-PAGE patterns of high molecular weight
(HMW) glutenins of the varieties Augusta and Hillsdale. Degradation of these
protein subunits began in the Augusta samples after two days of germination. At
the same time, several bands below the HMW glutenins became darker. By day
five of germination, almost complete loss of the HMW glutenins had occurred in

the Augusta grain.

Similar to the Augusta HMW glutenin bands, the band intensity in the
Hillsdale gel appeared to decrease after two days of germination. However,
throughout the complete period of germination, evidence of HMW glutenin
subunits remained. Similar to the Augusta gel, the Hillsdale gel also showed
bands in the lower portion of the gel which became darker, as germination

progressed.

Unreduced protein extracts, from the cultivars Augusta and Hillsdale,
examined using SDS-PAGE, are shown in Figure 20. Protein too large to enter
the gel can be seen at the top of both the Augusta and Hillsdale gels. However,
after one day of germination of the Augusta grain, a loss in the intensity of the
stain at the top of this gel can be seen. This loss continues throughout the

period of germination, but evidence for protein remains up to and including day

772

Fig. 19. SDS-PAGE of reduced HMW-glutenins from the cultivars Augusta
and Hillsdale at various stages of germination. A, cultivar Augusta; H,
cultivar Hillsdale; lane 1, ungerminated; lane 2, 24 h soak; lanes 3, 4, 5,6,
and 7, 1 through 5 days of germination, respectively; NP, standard
Neepawa; arrows, protein bands appearing during germination. Eight
microliters of protein extract loaded for NP and twelve for each of the other
samples.

 

773

i
l
‘0.
I
I
l
.l

 

99' ZIdN

l

774

Fig. 20. SDS-PAGE of unreduced HMW-glutenins from the cultivars
Augusta and Hillsdale at various stages of germination. A, cultivar
Augusta; H, cultivarHillsdale; lane 1, ungerminated; lane 2, 24 h soak;
lanes 3, 4, 5, 6, and 7, 1 through 5 days of germination, respectively. Twelve
microliters of protein extract were loaded for each sample.

 

775

 

776

five of germination. The gel containing unreduced protein from Hillsdale

samples displays no loss of the protein which did not enter the gel.

Gliadin proteins from the Augusta and Hillsdale treatments are shown in
Figure 21. There appeared to be some degradation of these proteins in the
Augusta kernels after three days of germination. From day three to day five,

however, there was apparently little change.

In contrast to the Augusta gliadin gel, the Hillsdale gel seems to have
maintained the same band intensity during the five days of germination. As
found for the Augusta grain, a few new bands appeared in the Hillsdale acid-

PAGE gel during germination.
DISCUSSION

Intervarietal differences in proteolytic activity during germination have
been reported (Bushuk et al 1971). However, comparisons between the various
cultivars examined in the literature are difficult to make due to differences in
methodology. For example, Marsh (1988) reported a 1.5-fold increase in
proteinase activity of a NaCI extract of two-day germinated wheat. In this case,
proteinase activity was measured using azocasein at 25°C and pH 5.8. After
eight days of germination, Hwang and Bushuk (1973) found a 17-fold increase in
proteinase activity. A two-fold increase in proteinase activity was reported by
Lukow and Bushuk (1984) for two cultivars germinated for 70 h using azocasein
at pH 6.0 for 2 h at 40°C. In addition to the proteinase activity methods used
being different in the studies cited above, rates of germination for the various
cultivars used where not reported. Thus adding to the difficulty of making

comparisons between the results reported for these studies.

777

Fig. 21. Acid-PAGE of gliadin proteins from the cultivars Augusta and
Hillsdale at various stages of germination. A, cultivar Augusta; H, cultivar
Hillsdale; lane 1, ungerminated; lane 2, 24 h soak; lanes 3, 4, 5, 6, and 7, 1
through 5 days of germination, respectively; NP, standard Neepawa;
arrows, protein bands appearing during germination. Eight microliters of
protein extract loaded for NP and twelve for each of the other samples.

 

 

778

779

The two varieties used for the present study were chosen because of their
differences in susceptibility to PHS as supported by the germination index
results. Augusta which had limited dormancy had greater proteinase activity than
Hillsdale which possess relatively more dormancy. In addition, Chapter Three of
this dissertation reports differences found between the proteinase activity of
several field-sprouted soft wheat cultivars. The proteinase activity results in the
present study and those found in Chapter Three, indicate that genotypes with
little resistance to PHS, when exposed to PHS environmental conditions, will

have greater activity than those with less resistance.

The pH of the PAGE (with copolymerized substrate) incubation buffers
was chosen to approximate the pH optima of wheat proteinases as well as to
simulate several situations. These situations included the previously reported pH
optima of proteinases in sound wheat (pH 3.8), the pH of wheat endosperm (pH
5.4), and the pH of various soft wheat product doughs and batters (pH 6.5)

(McDonald and Chen 1964, Hoseney 1994)

The proteinases which were active, both in the quiescent and germinating
grain (B-1 and B-2), are also those which would be most likely to have their
activity enhanced during the processing of soft wheat products, such as cakes
and cookies (Fig. 11). Possessing pH Optima close to neutral suggests that
these bands contain serine and/or metallo-proteinases. However, this is not
supported by the inhibitor work: crude extracts of the ungerminated Augusta

displayed no inhibition by either 3,4 DIC or 1,10 PA. Possible explanations are

720

as follows: (1) either aspartic and/or cysteine proteinase with neutral pH optima
are present, (2) the enzymes are serine and/or metallo-proteinases which are
not inhibited by the inhibitors employed in this study, or (3) the serine and/or
cysteine proteinase activity was so low that the azocasein with inhibitor assays
were not sensitive to detect their presence. Aspartic proteinases in general have
acidic pH optima, but some, such as renin, act at neutral pH (Barrett 1986).
Similarly, cysteine proteinases tend to have acidic pH optima, however, pH
optima near seven are also known to exist (e.g., papain). Although 3,4 DIC is
said to be a general serine proteinase inhibitor due to its inhibition of many
trypsin-like, chymotrypsin-like, and elastase-like proteinases, it has also proven
to be ineffective at inhibiting serine proteinases such as papain (Harper and
Powers 1985). A similar situation exists for 1,10 PA as well. Thus both number
one and two stated above cannot be ruled out; however, examining previous

inhibitor work seems to help clarify the situation.

McDonald and Chen'(1964) reported that the proteinase activity of
ungerminated wheat was not affected by trypsin or chymotrypsin inhibitors, thus
suggesting that either serine proteinases were not present, or the reversible
protein inhibitors used lacked specificity for the serine proteinase present.
Evidence suggesting the presence of serine proteinase in wheat has been
provided by Belitz and Lynen (1974). These authors reported that proteinase
activity was inhibited by diisopropylfluorophosphate (DFP), a proteinase inhibitor

which shows little nonserine proteinase inhibition. However, DFP has also been

727

reported to inhibit plant carboxypeptidases which are known to exist in
ungerminated grain (Kruger and Preston 1977). Salgo (1981) has suggested
that metallo-proteinases are present in wheat. However, this support was weak
at best because only the nonspecific proteinase inhibitor EDTA was used to
base this conclusion on. Recently, the first persuasive evidence for the
presence of metallo-proteinases and serine proteinases in barley seed has been
reported (Wrobel and Jones 1992). Initially using a crude extract separated with
PAGE gel copolymerized with gelatin, these proteinases appeared in the same
region as B-1 and B-2 in the present study. The activity in ungerminated barley
was reported to very low, but increased with germination. Results from serine or
metalIo-proteinase inhibitor studies were not reported for the ungerminated
barley. Further work by these authors included separating the extract from barley
germinated for four days, using carboxymethyl cellulose chromatography, and
then, with the PAGE system (Wrobel and Jones 1993). Five serine and five
metalIo-proteinases which were identified using phenylmethylsulfonyl fluoride
(serine proteinase inhibitor), DFP, EDTA, and dithiothreitol (a metallo-proteinase
inhibitor) possessed a close to neutral pH optimum. If it is assumed that the
neutral enzymes, in the four day germinated barley, are the same as the
enzymes in the ungerminated barley, then it would appear that serine and
metalIo-proteinases are present in ungerminated barley. As a result, it seems
possible that B-1 and B-2 from ungerminated soft wheat contain serine and/or

metallo-proteinases. Perhaps the inhibitors used only showed significant

722

inhibition after an increase in activity was noted for B-1 and B-2 during
germination, because, as suggested above, the serine and/or metallo-proteinase

activities were too low to measure in the ungerminated wheat.

Along with evidence for serine and/or metallo-proteinase in ungerminated
soft wheat discussed above, the inhibitor studies added support to previous
work reporting cysteine (Skupin and Warchalewski 1971, McDonald and Chen
1964) and aspartic proteinases (Lin et al 1993, Dunaevskii et al 1990,

Kawamura and Yonezawa 1982) in sound wheat.

Regarding the work by Lin et al (1993), it should be mentioned that the
aspartic proteinase from soft wheat, partially purified by these authors, had a pH
optimum of 4.1. In the present study, no activity was visualized using gelatin as
the substrate at pH 3.8 in the quiescent grain. However, using azocasein in the
activity assay did show proteinase activity, which was inhibited by an aspartic
proteinase inhibitor found to be present in the quiescent wheat samples. This
certainly furnishes evidence that substrate specificities exist for wheat

proteinases.

The initiation of HMW glutenin subunit mobilization coincided with the
increase in proteinase activity determined using the azocasein assay, as well as
with the first proteinase activity bands seen on the native PAGE Augusta gel
incubated at pH 3.8. These proteinases span a rather large range in mobility on
a native PAGE gel, and their acidic pH optima suggest that these bands contain

aspartic and/or cysteine proteinase. An interesting question raised by the gel

723

mentioned above is, what is the role of the germinative proteinases present in A-
2D, but not in A-3D (B-9 and B-10)? Mayer and Shain (1974) have suggested
that proteinase present early in the germination process may have the regulatory
function of activating or releasing bound enzymes. Quiescent wheat is known to
have cysteine proteinases which make up a large portion of its total proteinase
activity and are associated with storage proteins (Kaminski and Bushuk 1969,
McDonald and Chen 1964). Perhaps the enzymes in B-9 and B-10 serve to
release these cysteine proteinases. This would agree with the finding in this
study that cysteine proteinase activity peaks near the second day of
germination. Another possibility would be that these proteinases may also have
the task of cleaving bonds in proteins, making the proteins better substrates for
other proteases present. Thus, these enzymes would only be needed during the

early stages of storage protein degradation.

Concomitant, with the largest increase in proteinase activity found using
the azocasein assay, between day one and two of germination, was the
appearance of the germinative enzymes from the cultivar Augusta on the PAGE
gel incubated at pH 3.8, and the beginnings of storage protein mobilization. The
inhibitor studies demonstrated that during this time aspartic proteinases are at
their lowest point of activity, cysteine proteinase are at their highest level, and
serine and metallo-proteinase activities are increasing. Thus, as it has been

previously suggested, cysteine proteinases appear to be the “work-horses" of

724

storage protein degradation, beginning some time near the second day of

germination (Shutov and Vaintraub 1987).

The role of the aspartic proteinases present in germinating grain appears
to be much more controversial. Some have suggested that aspartic proteinases
initiate storage protein mobilization prior to the de novo biosynthesis of cysteine
proteinases (Doi et al 1980, Mikola 1983). Contrarily, Shutov and Vaintraub
(1987) have indicated that aspartic proteinases may quicken protein degradation
after it is initiated by cysteine proteinases. Recently, a barley grain aspartic
proteinase has been found to be related to mammalian lysosomal cathepsin D,
which is known to regulate the activity of other enzymes by cleavage of
regulatory enzymes (Sarkkinen et al 1992). Thus, the authors have suggested a

similar role for aspartic proteinase in cereal grains.

The inhibitor studies on extract from germinating soft wheat in the present
investigation, lend support to the findings of Wrobel and Jones (1993, 1992);
that is, that serine and metallo-proteinase are present in germinating cereal
grains. These authors reported having evidence of one EDTA-inhibited
proteinase after one day of germination, which is approximately the same time
that the present study using 1,10 PA and 2,3 DIC demonstrated evidence for

serine and metalIo-proteinase in soft wheat.

The gel containing unreduced Augusta proteins (Fig. 20) suggests that
after one day of germination, reduction of glutenins begins. This correlates with

work by Kobrehel et al (1992) which found glutenin as well as gliadin reduction

725

in a durum wheat cultivar began after one day of germination. This reduction
peaked after two to three days of germination with glutenins experiencing a five-
fold increase in reduction and gliadins a two-fold increase. Interestingly,
evidence of unreduced glutenins remained even up to five days of germination
of Augusta. Why the cultivar Hillsdale appeared to have no loss of unreduced

glutenins as seen at the top of the SDS-PAGE gel in Figure 20 is unclear.

A question raised by this study is why the HMW glutenins are apparently
cleaved earlier than the gliadins. Are there proteinases whose substrate
specificities include only the gliadins, or the glutenins, or is the structure of the
glutenins more suited to attack by the germination proteinase? Previous work by
Masson et al (1986) suggests the latter may be the case. Their work
demonstrated that glutenin was more rapidly hydrolyzed by porcine pepsin, a
serine proteinase, than was gliadin. Also, the digests of each were composed of
polypeptides differing in molecular weight, amino acid composition, and surface
hydrophobicity, thus confirming previous work by Bietz and Rothfus (1970).
Masson et al (1986) suggested that differences in peptic cleavage site location,
that Is, differences in site accessibility in gliadins versus glutenins, could explain

these results.

Previous work has suggested that once initially cleaved during
germination, storage proteins quickly are reduced to amino acids with little time
spent as smaller proteins or peptides (Preston et al 1978, Coulson and Sim

1965). Although this may be true in terms of relative quantities of protein

726

degraded, the present study does demonstrate that small proteins, i.e., those
with faster mobility on SDS-PAGE and Acid-PAGE gels (see Fig. 19 and 21), are
formed during the germination process presumably as a result of the cleavage of
larger proteins. Lukow and Bushuk (1984) reported an increase in intensity of
several reduced gliadin bands during germination of wheat, while Ariyama and
Khan (1990) found no changes in reduced or unreduced gliadin bands after
germination. Attempting to resolve the apparent disagreement between these
studies, Ariyama and Khan (1990) suggested that divergences may have
resulted from differences in varieties or germination procedure used. The former
suggestion would agree with the present study which demonstrates varietal

differences in the effect of germination on storage proteins.

Little can be said regarding the differences noted for the PAGE gels with
copolymerized gelatin, loaded with extract from the cultivar Augusta versus
Hillsdale as well as the differences found in germination influence on these
cultivars storage proteins. It could be assumed that the lower proteinase activity
found for the cultivar Hillsdale resulted in such investigations as B-9 and B-10
not being evident on the Hillsdale proteinase gel incubated at pH 3.8, no signs
of Hillsdale gliadin protein mobilization, and apparently, no change in the
amount of unreduced glutenins during five days of germination. However, it is
also possible that the presence of different proteinase isoenzymes or differences
in the structure of the storage proteins played a role in the differences noted

above.

727

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Masson. P., Tomé, D. and Popineau, Y. 1986. Peptic hydrolysis of gluten,
glutenin, and gliadin from wheat grain: kinetics and characterization
of peptides. J. Sci. Food Agric. 3721223.

Mayer, AM. and Shain, Y. 1974. Control of seed germination. Annu. Rev.
Plant Physiol. 25:167.

McDonald, CE. and Chen, LL. 1964. Properties of wheat flour proteinases.
Cereal Chem. 41:443.

Mikola, J.1983. Proteinases, peptidases and inhibitors of endogenous
proteinases in germinating seeds. In: Seed Proteins. J. Daussant, J.
Mosse, and J. Vaughan (eds). Academic Press, NY. pp. 35-52.

Ng, P.K.W. and Bushuk, W. 1987. Glutenin of Marquis wheat as a reference for
estimating molecular weights of glutenin subunits by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. Cereal Chem. 64: 324.

Ng, P.K.W. and Bushuk, W. 1990. Genetic origin of a unique gliadin doublet of
Thatcher-type bread wheats. Can. J. Plant Sci. 70:303.

Pizzinatto, A. and Hoseney, R. C. 1980. Rheological changes in cracker
sponges during fermentation. Cereal Chem. 57:185.

729

Preston, K. R., Dexter, J. E., and Kruger, J.E. 1978. Relationship of
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Salgo, A. 1981. Study of wheat proteases. Ph.D. dissertation. Technical
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Sarkkinen, P., Kalkkinen, N., Tilgmann, C., Siuro, J., Kervinen, J., and Mikola, L.
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Wrobel, R. and Jones, BL. 1993. Identification and partial characterization of
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Wrobel, R. and Jones, 8. L. 1992. Appearance of endoproteolytic enzymes
during the germination of barley. Plant Physiol. 100: 1508.

CHAPTER FIVE

CONCLUSIONS

1. Throughout kernel maturation, proteinase activity was significantly higher in

the field-grown grain (FGG) compared to the greenhouse-grown grain (GHG).

2. Alpha-amylase activity was significantly higher in the FGG compared to the
GHG at 21 and 28 days post anthesis (DPA), the same at 35 DPA, and greater

at 42 DPA.

3. Considering all stages of growth, proteinase activity was positively correlated
(r = 0.875, p<0.01) with alpha-amylase activity. The protein content of the grain
was highly correlated (r=0.930, p<0.01) with the growing conditions (i.e., higher
in the GHG compared to the FGG). However, the correlation with proteinase
and alpha-amylase activities was much lower (r=0.287, p<0.01; and r=-0.310,

p<0.01).

4. At harvest maturity, the correlation between proteinase and alpha-amylase
activity in the FGG, was weaker (r = 0.669, p<0.05) than the relationship in the
grain during maturation. Also, germination index was correlated with proteinase

(r = 0.553, p<0.05) and alpha-amylase activities (r = 0.622, p<0.05).

130

737
5. The cultivars Hillsdale and P2548 grown in the field and harvested at 42 DPA

had significantly less proteinase activity than the other seven cultivars, while
Augusta and Lowell had significantly more. The alpha-amylase activity found for

the cultivar Lowell was significantly higher than for all other cultivars.

6. In the laboratory-germinated grain, Augusta, the variety which had a greater
germination index, displayed greater proteinase activity than Hillsdale, the

variety with more dormancy.

7. Using class-specific proteinase inhibitors, soft winter wheat was found to
contain cysteine and aspartic proteinase activity during kernel maturation. The
cysteine proteolytic activity of aquatic extracts decreased as kernels matured,

while the aspartic proteolytic activity increased.

8. Through the use of native polyacrylamide gel electrophoresis (PAGE) with
copolymerized gelatin, it also appears likely that mature soft winter wheat

contains serine and/0r metallo-proteinase.

9. Soft winter wheat aspartic proteinase activity declined as a percentage of total
activity from the quiescent stage to the second day of germination; this activity
then increased, ending after five days of germination at a lower level than in the

quiescent kernel.

10. Soft winter wheat cysteine proteinase activity increased as a percentage of
total activity from the quiescent stage to the second day of germination; this

activity then decreased until five days of germination. The cysteine proteinases

732

thus appear to be those responsible for the early stages of storage protein

mobilization.

11. Beginning after one day of germination, serine and metallo-proteinase
activity began to increase as a percentage of total proteinase activity in
germinating soft winter wheat; these activities peaked after four days of

germination.

12. Ten bands of proteinase activity were visualized using native PAGE
copolymerized with gelatin and crude extracts from quiescent and germinating
Augusta kernels. Two bands were present in quiescent grain at close to neutral
pH, thus suggesting that the bands contain metallo and/or serine proteinase.
The other eight zones which were present during germination had acidic pH

optima, indicating that they contain either aspartic or cysteine proteinase.

13. The glutenin storage proteins in germinating soft winter wheat appeared to

be degraded sooner during germination than the gliadins.

CHAPTER SIX

FUTURE WORK

Much remains to be understood about the proteinases in wheat.
Answering the questions still unanswered rests on initially purifying and
characterizing each of the classes of proteinases found in wheat during the
present study. Determining their location in the kernel during the various stages
of the life cycle would then lead to understanding the role(s) these enzymes play
in maturing and germinating wheat. With this information in hand, various
applications for the food industry could arise which could be exploited by using
the tools of biotechnology to produce wheat proteinases. A specific example that
comes to mind is saltine cracker production. Endogenous proteolytic enzymes in
flour are thought to be important to the desired changes the rheological
properties of cracker doughs undergo during fermentation. Although commercial
preparations of fungal and bacterial proteolytic enzymes have been used to try
to reduce fermentation times during cracker processing, none have been
completely successful. This situation could perhaps be improved with the
availability of proteinases whose substrate specificities include the wheat

proteins which are cleaved during cracker dough fermentation.

133

734

Having determined in this study that genotypic differences exist for
proteinase activity in preharvest sprouted grain, a next step would be to
determine the effect of these differences on the quality of various soft wheat

products.

APPENDIX A

Varieties Used to Investigate Enzyme Activity in Soft Winter Wheat

 

 

Genotype Pedigree Grain Color OLigin
Cardinal Virginia 635212 x Logan/l Red Ohio
Blue boy x (Logan x 2)
Hillsdale Asosan/Genesee *4// Red Michigan
VA 66-54-10
Twain Knox 62/SRW 14-74 Red Indiana
Mendon (GeneseeANinoka,xo467)/5/ Red Michigan

(B2141, (Suwon 92/Brevor/2/
5*Genesee, A6506)/4I(A4528,
Norin 10lBrevorl2/Yorkwin/3/
Genesee*4/2/Norin
10/Brevor))

P2548 Hadden*2/4/Georgia 1123 /3/ Red Indiana
Norin 10/Brevor/2/Tenmarq/5/
(Vigo/Clarkan/2/Norin 66,
M06582/3/Redcoat/6/Coker
68-15/5/(M07910, Etoile de
Choisy/2/Thorne/Clarkan/4/
Pawnee/3/(Pd3848A5-5-26,
Citr12454, Trumbull/W38/2l

Fultleungarian))

Augusta GeneseelRedcoat 82747// White Michigan
Yorkstar

Frankenmuth Norin 10/Brevor 14l/Yorkwin/ White Michigan

3/2 *Genesee A3141/4/
Genesse *3/Redcoat A5115

Geneva Burt/5/Genesee/4/Frondosol3/ White New York
Trumbull/lHope/Hussa R/6/
Ross/7/Genesee

Lowell (GeneseeNVinoka,xo467)/5/ Wh'te Michigan

(B2141, (Suwon 92/Brevorl2/
5*Genesee, A6506)/4/(A4528,
Norin 10/Brevorl2/Yorkwin/3/
Genesee*4/2/Norin
10/Brevor))

135

APPENDIX B
Tabulated Data

Moisture Content of Soft Winter Wheat Grown in Dansville, Michigan, in 1992

 

 

 

Wheat Moisture (%)

Cultivar 21 DPAa 28 DPA 35 DPA 42 DPA
Mk
Augusta 59.27 44.98 39.66 32.39
Frankenmuth 58.99 47.08 42.50 32.66
Geneva 55.14 49.05 41.03 33.46
Lowell 57.99 50.09 39.38 23.38
@1
Cardinal 56.89 46.29 40.11 30.60
Hillsdale 56.11 43.56 41.08 30.90
Mendon 57.27 51.75 40.40 30.93
P2548 55.81 47.06 38.34 25.56
Twain 58.84 48.92 42.96 27.21

 

3 Days post anthesis

136

737

Moisture Content of Soft Winter Wheat Grown in a Greenhouse

 

 

 

Wheat Moisture Q/o)

Cultivar 21 DPAa 28 DPA 35 DPA 42 DPA
Mlle
Augusta 52.36 ‘ 43.43 18.99 11.51
Frankenmuth 50.37 43.96 18.61 10.60
Geneva 53.98 44.56 21.93 11.85
Lowell 51.56 47.45 27.56 ‘ 11.82
Reg
Cardinal 51.55 43.22 26.29 11.80
Hillsdale 48.20 44.08 21.68 11.28
Mendon 50.81 46.12 34.14 11.32
P2548 56.72 48.21 26.53 11.08
Twain 56.37 47.88 30.77 11.74

 

3 Days post anthesis

738

Protein Content of Soft Winter Wheat Grown in Dansville, Michigan, in 1992

 

 

 

Wheat Protein (%)°

Cultivar 21 DPAb 28 DPA 35 DPA 42 DPA
wme.
Augusta 9.58 9.74 10.28 10.32
Frankenmuth 10.18 10.23 10.51 10.76
Geneva 11.03 11.89 11.42 11.98
Lowell 10.02 9.98 9.50 9.94
B_e_d_
Cardinal 9.43 9.79 10.28 10.73
Hillsdale 10.23 9.99 11.64 10.00
Mendon 10.21 9.78 9.55 9.77
P2548 9.14 9.67 9.71 10.12

' Twain 10.46 12.05 11.66 11.69

 

a Values are means on a dry wt basis

'3 Days post anthesis

739

Protein Content of Soft Winter Wheat Grown in a Greenhouse

 

 

 

Wheat Protein (%)a

Cultivar 21 DPAb 28 DPA 35 DPA 42 DPA
W_hit§
Augusta 14.81 15.49 17.39 16.87
Frankenmuth 15.22 15.64 17.53 17.65
Geneva 15.43 16.59 17.43 17.54
Lowell 16.44 17.37 18.17 18.12
Bed
Cardinal 17.54 16.56 18.19 18.31
Hillsdale 14.34 16.22 18.04 18.07
Mendon 16.94 18.66 19.36 18.88
P2548 14.67 14.80 16.01 17.04
Twain 19.29 20.25 20.62 21.14

 

a Values are means on a dry wt basis
b Days post anthesis

740

Alpha-Amylase Activity in Soft Winter Wheat Grown in Dansville, Michigan, in
1992

 

 

 

Wheat Alpha-Amylase Activity
(DU)a,b

Cultivar 21 DPAc 28 DPA 35 DPA 42 DPA
MILE:
Augusta 1.84 0.52 0.09 0.38
Frankenmuth 2.05 0.63 0.09 0.08
Geneva 1.46 0.45 0.13 0.29
Lowell 1.03 0.34 0.08 1.31
_R_e_g
Cardinal 1.32 0.46 0.10 0.11
Hillsdale 1.87 0.57 0.13 0.07
Mendon 1.47 0.45 0.07 0.23
P2548 1.08 0.34 0.07 0.06
Twain 1.33 0.33 0.19 0.15

 

a Dextrinizing Units
b Values are means on a dry wt basis

° Days post anthesis

747

Alpha-Amylase Activity in Soft Winter Wheat Grown in 8 Greenhouse

 

 

 

Wheat Alpha-Amylase Activity
(DUfb

Cultivar 21 DPA“ 28 DPA 35 DPA 42 DPA
Wilts
Augusta 0.74 0.15 0.10 0.11
Frankenmuth 0.78 0.17 0.10 0.10
Geneva 1.28 0.26 0.11 0.11
Lowell 0.81 0.21 0.10 0.09
Reg
Cardinal 0.71 0.23 0.10 0.10
Hillsdale 0.67 0.16 0.10 0.10
Mendon 0.77 0.27 0.16 0.09
P2548 1.03 0.30 0.08 0.09
Twain 1.16 0.40 0.13 0.16

 

a Dextrinizing Units
'3 Values are means on a dry wt basis

° Days post anthesis

742

Proteinase Activity in Soft Winter Wheat Grown in Dansville, Michigan, in 1992

 

 

 

Wheat Proteinase Activity 31"

Cultivar 21 DPAc 28 DPA 35 DPA 42 DPA
_W_h_it_e.
Augusta 3.451 1.546 1.233 1.089
Frankenmuth 3.218 1.478 1.210 0.942
Geneva 3.647 1.629 1.064 0.948
Lowell 3.018 1.401 1.137 1.049
R_ad
Cardinal 2.733 1.493 1.026 0.899
Hillsdale 3.895 1.574 1.067 0.909
Mendon 3.239 1.382 1.135 0.962
P2548 3.262 1.387 1.096 0.898
Twain 2.873 1.389 1.292 0.983

 

a Absorbance at 366 nm
b Values are means on a dry weight basis

° Days post anthesis

743

Proteinase Activity in Soft Winter Wheat Grown in a Greenhouse

 

 

 

Wheat Proteinase Activity 3'”

Cultivar 21 DPAc 28 DPA 35 DPA 42 DPA
M112
Augusta 2.479 1.258 0.900 0.722
Frankenmuth 2.325 1.210 0.824 0.719
Geneva 2.928 1.324 0.999 0.771
Lowell 2.672 1.330 0.909 0.742
B_e_q
Cardinal 2.285 1.300 0.981 0.788
Hillsdale 2.152 1.275 0.908 0.695
Mendon 2.615 1.457 1.088 0.787
P2548 2.585 1.476 0.957 0.782
Twain 2.371 1.335 0.999 0.757

 

a Absorbance at 366 nm
b Values are means on a dry weight basis

° Days post anthesis

744

Germination Index for Soft Winter Wheat Grown in Dansville, Michigan, in 1992

 

 

Wheat Germination lndexa

Cultivar 21 DPAb 28 DPA 35 DPA 42 DPA
white
Augusta 0 10 70 290
Frankenmuth 0 0 7 257
Geneva 4 14 60 308
Lowell 3 1 55 313
lied
Cardinal 0 1 34 200
Hillsdale 0 0 2 82
Mendon 0 1 50 235
P2548 0 0 8 91
Twain 0 0 2 95

 

a Germination Index is the number of kernels which germinated during 5 days, with
greater weight given to those germinating earlier

b Days post anthesis

745

Germination Index for Greenhouse Grown Soft Winter Wheat

 

 

 

Wheat Germination Indexa

Cultivar 21 DPAb 28 DPA 35 DPA 42 DPA
White
Augusta 3 5 1 36
Frankenmuth 0 1 0 0
Geneva 0 1 2 1
Lowell 3 3 0 1
B_e_d
Cardinal 1 2 0 1
Hillsdale 0 1 0 0
Mendon 1 3 0 8
P2548 0 0 0 0
Twain 1 1 0 1

 

a Germination Index is the number of kernels which germinated during 5 days, with
greater weight given to those germinating earlier

b Days post anthesis

746

Analysis of variance of alpha-amylase activity in nine soft winter wheat cultivars
grown in Dansville, Michigan, 1992, and harvested at 42 days post anthesis

 

 

Source of Variance DF Sum of Squares Mean Square F-Ratio P
Variety 8 4.982 0.623 4.969 0.001
Block 1 1.210 1.210 9.656 0.005
Error 26 3.258 0.125

 

Analysis of variance of proteinase activity in nine soft winter wheat cultivars
grown in Dansville, Michigan, 1992, and harvested at 42 days post anthesis

 

 

Source of Variance DF Sum of Squares Mean Square F-Ratio P
Variety 8 0.121 0.015 10.385 0.000
Block 1 0.016 0.016 11.233 0.002

Error 26 0.038 0.001

 

APPENDIX C
Germination Index Formula

(Walker-Simmons 1987 - Chapter Three)
Germination Index = (5 x n1 + 4 x n2 + 3 x n3 + 2 x n4 +1 x n5)

Where n1, n2, n3, n4 and n5 are the number of seeds germinated on the first,
second and subsequent days until the fifth day, respectively. The numbers 5, 4,
3, 2 and 1 are the weights given to the number of seeds germinated on the first,
second and subsequent days, respectively.

SDS-PAGE Protocol
(Ng and Bushuk 1987 - Chapter Four)

Glutenin Extraction

Let sit at room temperature 40 g of sample plus 1 ml extraction buffer.
Vortex periodically. Heat in boiling water for 2.5 min. Aliquots of the
supernatant are used as the protein extract.

Extraction buffer stock solution

glycerol 20.0 ml
stacking-gel buffer (below) 12.5 ml
MilliQ water 24.1 ml
SDS 4.0 g
Pyronin Y 20.0 mg

Extraction buffer (made just prior to use)
MilliQ water 24.0 ml
extraction buffer stock solution 10.2 ml
mercaptoethanol 1.8 ml

147

748

Vertical electrOphoresis unit (140 mm x 160 mm x 1.5 mm)

Per Two Gels (ml)
Separating Gel

Acrylamide (35%) 49.30 0
Bisacrylamide (2%) 3.880
Separating-gel buffer (1 M Tris, pH 8.8) 37.600

(Degas)

SDS solution (10%) 1.000
Ammonium persulfate (1%) 2.500
TEMED 0.050

Stacking Gel

Acrylamide (35%) 1.710
Bisacrylamide (2%) 0.430
Stacking-gel buffer (1.0 M Tris, pH 6.8) 2.500
MilliQ water 14.400
(Degas)

Ammonium persulfate (1%) 0.750
N,N,N°,N'-Tetramethylethylenediamine (TEMED) 0.015

Running Buffer (pH 8.3)

Tris 24.228 9
Glycine 115.308 9

SDS solution (10%) 80 ml

MilliQ water complete to 8 L

Electrophoresis Running Conditions
8 ttl of sample extract
4°C
5 mA per gel for 2 h, then 10 mM per gel for 18 h, then 15 mM per gel
for 2 h

749

Gel Rinsing
Agitate gel in rinsing solution for 1 h, drain, repeat 2 times

Rinsing Solution
TCA (100%) 100 ml
Methanol 330 ml
MilliQ water 570 ml

750

Acid-PAGE Protocol
(Ng et al 1990 - Chapter Four)

Gliadin Extraction

100 mg of sample plus 200 ill of 70% ethanol, vortex briefly. Let sit at
room temperature for 15 min. Centrifuge for 2 min, at 10,000 x G, at room
temperature. Remove supernatant and add 1.25 times its volume with dilution
solution (0.25% w/v aluminum lactate, adjusted to pH 3.1 with lactic acid, 50%
wlv sucrose and 3% w/v methyl green).

(Lafiandra and Kasarda 1985 - Chapter Four)

Apparatus
Vertical electrophoresis unit (140 mm x 160 mm x 1.5 mm)

Solutions
A: Acrylamide (28%) and bisacrylamide (1.2%)
B: Potassium hydroxide (3.5 g) and lactic acid (25 ml of 85%) per 100 ml
C: Aluminum lactate (6.25 g) and lactic acid (10 ml of 85%) per 100 ml
D: Silver nitrate (17 mg per ml)
E: Ammonium persulfate (90 mg per 100 ml)
F: Hydrogen peroxide (100 ill of 30% H202 diluted to 3 ml)
G: Solution A (17 ml), solution B (2 ml), ascorbic acid (20mg), and
ferrous sulfate (2.5 mg) per 100 ml
Per Two Gels (ml)

Separating Gel
Solution A 20.00
Solution B 1.60
Solution 0 0.80
Solution E 40.00
MilliQ water complete to 80 ml

(Degas 2 min)

757

Stacking Gel
Solution G (Thaw) 10.000
Solution F (Degas) 0.015

Running Buffer
50-fold dilution of solution B 5 L

Electr0phoresis Conditions
Prerun stacking gels for 1 h at 4°C and 45 mM Amp per gel
Use 0.5 L running buffer in upper chamber and 4.5 L in the
lower chamber. Discard the upper chamber buffer after prerun.

After pouring stacking gel, rinse with solution C (diluted 50-fold and

adjusted to pH 3.1 with lactic acid)

Load 7-25 ttl aliquot of protein extract

752
PAGE with Copolymerized Substrate Protocol
(Wrobel and Jones 1992 -Chapter Four)

Apparatus
mini-gel with 1.5 mm spacers

Separating Gel Per Two Gels (ml)
Acrylamide (30%) and 7.420
bisacrylamide (1%)
1.5 M Tris buffer, pH 8.8 5.060
Substrate (1%) 2.020
MilliQ water 5.730
Ammonium Persulfate (10%) 0.045
(Degas)
TEMED 0.025
Stacking Gel
Acrylamide (30%) and 1.000
bisacrylamide (1%)
0.5 M Tris buffer, pH 6.8 1.250
MilliQ water 7.750
Ammonium persulfate (10%) 0.100
(Degas)
TEMED ' 0.050

Running Buffer
0.025 M Tris, 0.192 M Glycine, pH 8.5

753

Electrophoresis Running Conditions

25 ill of sample buffer (1 M Tris, pH 6.8, 2% sucrose, trace bromophenol
blue) and crude extract (1:1)

10 mA per gel, 4.5 h, at 4°C

Gel Incubation Conditions
0.1 M acetate buffer (pH 3.8), 2 mM cysteine,15 h, 40°C
0.1 M acetate buffer (pH 5.4), 2 mM cysteine,18 h, 40°C
0.1 M phosphate buffer (ph 6.5), 2 mM cysteine,18 h, 40°C

Gel Staining

1 h in 0.1% amido black in acetic acid2methanol2water (10:30:60)

Gel Destaining
6 h (3 changes) in acetic acidzmethanolzwater (10:30:60)

"IIIIIIIIIEIIIIIIIIIEEE