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ENZYMATIC AND METABOLIC CHANGES
IN THE ‘COTYLEDONS OF
THE BAMBARRA GROUNDNUT .
(VOANDZEIA SUBTERRANEA L. THOUARS)
DURING. GERMINAT'ION.

Thesis for the Degree of M. S.
MiCHIGAN STATE UNIVERSITY
KOFI AMUTI
. 1972

    
    

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Michigan State
Umvcrsity

    
 

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ABSTRACT

ENZYMATIC AND METABOLIC CHANGES IN THE
COTYLEDONS OF THE BAMBARRA GROUNDNUT
(VOANDZEIA SUBTERRANEA L. THOUARS)
DURING GERMINATION

 

BY

Kofi Amuti

The study reported here was designed to investigate
some metabolic and enzymatic changes which occur in the
cotyledons of dark-grown bambarra groundnut (Voandzeia sub-
terranea L. Thouars) seeds during the first eleven days of
germination. Two cultivars were used.

It was observed that the cotyledons of the dry
seeds possessed some activity of all of the enzymes studied
except peroxidase. The a-amylase, acid phosphatase, a-
galactosidase and peroxidase activities increased in the
cotyledonary tissue with time of germination. The a-
glucosidase and B-galactosidase activities increased for
the first five days and decreased thereafter. 8-
glucosidase showed a general pattern of decreased activity
with germination. The malate dehydrogenase of the mito-
chondria showed a slight increase with time while the

l

Kofi Amuti

activity in the post-mitochondrial fraction decreased dur-
ing the first five days of germination before increasing
and then decreasing subsequently by the eleventh day. The
total malate dehydrogenase reflected that of the post-
mitochondrial fraction.

The carbon dioxide evolution, oxygen uptake and
respiratory quotient (RQ) values increased with the length
of imbibition period over the first twenty-one hours.

The growth regulators, gibberellic acid and benzyla-
denine, had varying effects on the enzymes studied in the
germinating bambarra groundnut cotyledons. Benzyladenine
delayed the increase in activity of a-amylase, peroxidase
and acid phosphatase but had no effect on a-galactosidase,
a- and B-glucosidases; while gibberellic acid increased
the activity of a—amylase and peroxidase but had no effect
on acid phosphatase, a-galactosidase and a- and
B-glucosidases.

The total lipid, carbohydrate and protein content
of the dark-grown bambarra groundnut cotyledons decreased
during germination. There was a slight overall decrease
in percent lipid and percent total carbohydrate with age
while the percent soluble carbohydrate increased to a maxi-
mum on day seven and thereafter decreased. The protein

2

Kofi Amuti

showed a tendency to increase slightly in percentage with
age in one cultivar, whereas in the other it remained

largely unchanged.

ENZYMATIC AND METABOLIC CHANGES IN THE
COTYLEDONS OF THE BAMBARRA GROUNDNUT

(VOANDZEIA SUBTERRANEA L. THOUARS)

 

DURING GERMINATION

BY

Kofi Amuti

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements”
for the degree of
MASTER OF SCIENCE

Department of Botany and Plant Pathology

1972

ACKNOWLEDGMENT

I would like to express my sincere thanks to Dr.
Clifford J. Pollard, my major professor, for his guidance,
encouragement and criticisms throughout the course of this
study. Thanks are also extended to Dr. William G. Fields
and Dr. Robert C. Herner, my guidance committee members.

I also wish to express my sincere thanks to Dr.
Suppiah Sinnadurai, Department of Crop Science, University
of Ghana, Mr. Nico Kwaku Ankudey of the Mole Game Reserve,
Ghana, and the Food and Agriculture Division of the
U.S.A.I.D. Mission to Ghana for making the bambarra
groundnut seeds available for this research.

Appreciation is given to the University of Ghana

for a Post-graduate Scholarship.

ii

TABLE OF CONTENTS

LIST OF TABLES O O O O O O O O O O O 0 O O O 0

LIST OF FIGURES O O O O O O O O O O O O O O 0

INTRODUCTION 0 O O O O O O O O O O O O O O O 0

Metabolic and enzymatic changes in seeds
during germination . . . . . . . . . .

Effects of growth regulators on enzymatic
changes in germinating seeds . . . . .

Bambarra groundnut (Voandzeia subterranea)

 

Literature review. . . . . . . . . . .

MATERIALS AND METHODS . . . . . . . . . . . .

Source and preparation of plant material.
Preparation of extract for enzyme assays.
Enzyme assays . . . . . . . . . . . . . .
Lipid, protein, and carbohydrate analyses
Respiration measurement . . . . . . . . .

Studies with growth regulators. . . . . .

RESULTS 0 O O O O I O O O O O O O O O O O O I

DISCUSSIONS O O O O O O O O O O O O O O O O 0

iii

Page

vi

10
10
11
12
14
15
15
16

37

TABLE OF CONTENTS (continued)

Page
APPENDIX 0 O O 0 O O O O O O O O O O O O O O O O O O 43
LITERATURE CITED 0 O O O O O 0 O O O O O O O O O O O 47

iv

LIST OF TABLES

Table Page

1. The effect of the growth regulators, giber-
ellic acid and N5-benzyladenine, on changes
in enzymatic activity of dark-germinated
cream/black 'eye' bambarra groundnut seed
between the 2nd and 10th day.. . . . . . . . 35

LIST OF FIGURES

Figure

1. Changes in a-amylase activity in the
cotyledons of dark-grown bambarra groundnut
seeds . . . . . . . . . . . . . . . . . . .

2. Peroxidase activity in the cotyledons of
dark-germinated bambarra groundnut seeds
(cream/black 'eye' cultivar). . . . . . . .

3. Changes in a-galactosidase activity with
time in bambarra groundnut cotyledons
grown in the dark . . . . . . . . . . . . .

4. Changes in the B-galactosidase activity in
the cotyledons of dark-germinated bambarra
groundnut seeds with time . . . . . . . . .

5. Pattern of a-glucosidase activity in the
cotyledons of cream/black 'eye' cultivar
germinated in the dark. . . . . . . . . . .

6. Pattern of B-glucosidase activity in the
cotyledons of dark-grown bambarra groundnut
seeds with time . . . . . . . . . . . . . .

7. Changes in the acid phosphatase activity in
the cotyledons of dark-grown bambarra
groundnut seeds with time . . . . . . . . .

8. Changes in malate dehydrogenase activity in
the mitochondria and post-mitochondria
fractions of the cotyledons of cream/no
'eye' bambarra groundnut seed with time . .

9. Pattern of O uptake, CO evolution and RQ
of the cotyledons of tfie cream/black 'eye'
bambarra groundnut with length of
imbibition. . . . . . . . . . . . . . . . .

vi

Page

17

18

20

21

22

23

24

25

27

LIST OF FIGURES (continued)

Figure

10.

ll.

12.

l3.

14.

15.

Changes in the fresh weight of the embryonic
axis of dark-grown bambarra groundnut
seeds. I O O O O O O O O O O O O O O O O 0

Change in percent lipid of the cotyledons
of the dark-germinated cream/black 'eye
bambarra groundnut seed with time. . . .

Changes in the percent protein of the dark-
grown bambarra groundnut seed cotyledons
With time 0 O O O O O O O O O O O O O O O 0

Changes in the percent total carbohydrates
of dark-grown bambarra groundnut seeds
with time. . . . . . . . . . . . . . . . .

Changes in the percent soluble carbohydrates
of dark-germinated cream/no 'eye' bambarra
groundnut with time. . . . . . . . . . . .

Changes in total carbohydrate, lipid and
protein contents with time of the COtyle-
dons of dark-germinated cream/black 'eye'
bambarra groundnut seeds . . . . . . . . .

vii

Page

28

29

31

32

33

34

ENZYMATIC AND METABOLIC CHANGES IN THE
COTYLEDONS OF THE BAMBARRA GROUNDNUT
(VOANDZEIA SUBTERRANEA L. THOUARS)
DURING GERMINATION

 

INTRODUCTION

Metabolic and enzymatic changes in seeds

during germination

 

The seed is the means of perpetuating the species
in seed-producing plants. At maturity, there are enough
stored materials in the cotyledons or endosperm to support
the develoPment of the embryo until the young plant begins
photosynthesis.

The germination process involves many changes in
the seed, the end result of which is the growth of the em-
bryo. One of the first observable changes during germina-
tion is the breakdown of the stored materials which results
in the products becoming available to the embryonic axis
(24). Such changes are brought about through the action

of the different enzymes in the seeds.

Some selected works related to that reported here
are as follow: In studies with germinating peanut (Arachis
hypogea) seeds, Rabari et al. (32) showed that there was no
synthesis of fatty acids and that the saturated acids were
metabolized faster than the unsaturated acids during germi-
nation. They also showed increases in sucrose, starch and
reducing sugar content at the expense of metabolized oil.
Cherry (5) showed that in the cotyledons of the germinating
peanut, 60% of the dry weight and 70% of the proteins were
depleted between four to nine days.

Because starch is one of the main storage materials
in a number of seeds, the changes in amylase in the seeds
during germination have been studied and the following find-
ings have been reported. It has been suggested that a-amylase
is involved in the initial degradation of starch reserves in
the cotyledons of germinating pea (17, 39). Kasugai (19)
observed an increase in the activity of this enzyme in the

cotyledons of the mung bean (Phaseolus aureus) with the

 

progress of germination and attributed the decrease in the
polysaccharides to its formation and action. In an earlier
study with different cultivars of beans, he observed the ac-
tivities of either or both B-amylase and a-amylase in the-
cotyledons (18). Other workers have reported increases in

a-amylase activity in germinating pea cotyledon; and the

Ill‘"||‘ull"

fact that this increase is the result of net enzyme protein
synthesis in the pea and d3 £939 synthesis in the barley
has been documented (ll, 39, 44).

With regard to the glycosidases, the following have
been reported: A decrease in the a-glucosidase and an in-
crease in the B-glucosidase activities, with increasing age
of the tissue, have been observed in the hypocotyl tissue

of Red Kidney beans (Phaseolus vulgaris) (28). An a-

 

glucosidase (maltase) has been suggested to hydrolyse the
maltose produced in the germinating pea cotyledon (38).

In alfalfa (Medicago sativa) seed, B-glucosidase and also

 

B-galactosidase are thought to be involved in the "reorgan-
ization of the storage glycoproteins and structural poly-
saccharides as an early event in germination following the
uptake of water" (33).

A decrease in activity of a- and B-galactosidase
with age of the hypocotyl tissue of the Red Kidney bean
has been reported (28L Compared with the cotyledons left

attached to the plant axis, exised cotyledons of Phaseolus

 

vulgaris showed an increase in a-galactosidase activity
which was shown not to be due to synthesis of enzyme pro-
tein (20). Though there were cultivar differences, coffee

seed showed an increase in the already high a-galactosidase

‘A—u-

activity upon soaking and subsequent germination (34).
Germinating cotton seeds have also been reported to show
increases in a-galactosidase activity (35).

An increase in phosphatase which was specific for
ADP and ATP and activated by cadmium was observed in the
seeds of germinating pea and this was thought to represent
net enzyme synthesis (44). Using the inhibitor azetidine-
2-carboxylic acid, a proline analog, Presley and Fowden
(31) obtained evidence that the increase in acid phosphat-
ase activity in the cotyledons of germinating peas, mung
beans, cucumber and sunflower was due to the activation of
zymogens and not to de_ngvg synthesis. The activity of
this enzyme has been observed to increase during the early
stages and decrease during the latter stages of germination

in dwarf beans (Phaseolus vulgaris) (12).

 

The cotyledons as well as roots of Phaseolus mungo

 

showed a steep increase in peroxidase activity beginning
at the fourth day of germination while the epicotyl and
hypocotyl were observed to maintain a low and almost con-
stant activity (13).

In germinating peanut cotyledon, the mitochondria
seem to increase in structure and internal organization and

later swell and disintegrate with the age of tissue. Also

there is an increase in some of the mitochondrial enzymes
followed by a rapid decrease thereafter while others in-
crease continuously with age of the seedling (4). A de—
cline in the q02 and P/O values has been observed in the
senescing castor bean endosperm (l). Malate dehydrogenase
activity in the mitochondrial fraction of the pea cotyledon
has been shown to increase during the imbibition phase of
germination while in the dry seed the post-mitochondrial
fraction has a significant activity (27).

In some peripheral studies with the senescing cor-

olla of Morning glory (Ipomea purpurea), a dramatic increase

 

in B-glucosidase with only slight increases in the acid
phosphatase and a-glucosidase has been reported. It was
also shown that protein synthesis was required for the in-

crease in the B-glucosidase and some other enzymes (23).

Effects of growth regulators on enzymatic

changes in germinating seeds

It has been observed in germinating pea cotyledons
that some factor or factors from the plant axis control

the events occurring in the cotyledons (45). It is such

observations and many others similar to it which have, I
think, stimulated the research into the effects of the
different growth regulators on plant tissue. It has been
reported that the pea cotyledons excised before 48 hours

of imbibition and treated with gibberellin (GA) and kinetin
had increased a-amylase activity. But those cotyledons ex-
cised before 48 hours of imbibition and not treated with
the growth regulators had low activity of this enzyme;
phosphatase activity was not developed in the treated and
untreated cotyledons excised before forty-eight hours imbi-
bition while those excised after imbibition for this length
of time had normal activity (41).

Treatment of barley aleurone layers with gibberellic
acid (GA) has been reported to result in the de'ngzg'synthe-
sis of many hydrolytic enzymes. It was also shown that in-
creasing concentrations of the GA caused the release of in-
creasing quantities of reducing sugars and proteins and a
prOportional decrease in the endosperm dry weight (2, 6, 29,
30, 42). Imbibing pea seeds in N6-benzy1adenine (BA), how-
ever, has been reported to cause a delay in the production
of amylase and starch degradation (1?, 37). The application
of BA to the seeds of the dwarf pea has been shown to result

in the delay of the cotyledon senescense. It induces

 

II .|‘.c‘|l.ll Ii 1

branching and is thought to alter the normal distribution
of nutrients between the cotyledons, stem and roots (36).
Gukova and Faustovo (14) are of the opinion that
GA treatment decreases the peroxidase activity in plants
but Michniewicz and Stanislawski (26) think there is in-

creased activity with GA treatment.

Bambarra groundnut (Voandzeia subterranea):

 

Literature review

 

The bambarra groundnut (Voandzeia subterranea L.

 

Thouars), a tropical pulse, is the only species in the
tribe Phaseoleae of the family Papilionaceae (15). Like

the peanut (Arachis hypogea), it produces its seeds under

 

the surface of the soil. The plant is said to be found in
the wild in Central Africa, in N.E. Nigeria, and in N.
Cameroun (8, 16). It has bunched, semi-bunched, or open
growth habit with trifoliate leaves which are carried on
long slender petioles. The flowers are normally carried

in pairs on short peduncles. Some species of ants are said
to be pollinators in Ghana though some cultivars are said

to be self-pollinated (8, 9, 15, 16). The crop is usually

 

, —r- .— qfl—au-r-h-w- m—‘-q.-

grown for home consumption and only the surplus is sold on
local markets (15, 16).

The young fruits and mature seeds, unlike peanuts,
have to be boiled before eaten. In some places, the ripe
seeds are said to be pounded into flour or soaked and
boiled into porridge and used as food on long journeys
(15, 16). In Ghana the young ripe fruit or mature seeds
are boiled and served with fried plantains.

The bambarra groundnut, though not rich in protein,
has oil and carbohydrate content which gives it a good
caloric value and make it a well balanced food. It is
sometimes preferred to peanuts (8, 16). Johnson (16)
quotes an ether extract value (lipid content) for the bam-
barra groundnut which is less than that for the peanut and
soyabean but higher than that of ration beans and cowpeas;
the crude protein content is the lowest for all the legumes
mentioned but its carbohydrate content is highest of all
(16). Value of 6.9, 20.1 and 60.0 percent for lipid, pro-
tein and carbohydrate content, respectively, have been

given (3). In comparison with groundnut (Arachis hypogea)

 

and COWpea (Vigna spp), Hepper (15) showed that the bambarra
groundnut has a protein content lower than that of the other

two legumes and an oil content higher than the COWpea but

| l I! [I J .l‘l

 

lower than the groundnut. He also cited the work of some
researchers who claim the leaves of the plant are also
good for fodder (15).

From the preceding, it is evident that some inves-
tigations have been conducted into the metabolic and enzy-
matic changes in the cotyledons and other tissues of germi-
nating seeds of some legumes. But no detailed study, to my
knowledge, has been conducted on the bambarra groundnut
seed. Thus this thesis research was designed to study and
document enzymatic and metabolic changes in the cotyledons
of the bambarra groundnut (Voandzeia subterranea L. Thouars)

seeds during germination.

MATERIALS AND METHODS

Source and preparation of plant material

 

Seeds of two cultivars of bambarra groundnut

(Voandzeia subterranea L. Thouars) used in the experiments

 

were obtained from Dr. Suppaih Sinnadurai. The small-
seeded cultivar (cream/no 'eye') is from the Department
of CrOp Science, University of Ghana bambarra groundnut
collections and the large-seeded (cream/black 'eye') cul-
tivar was purchased from a local market in Ghana.

The seeds were planted in vermiculite in the dark
at 25°C and precautions were taken to exclude light since
it was observed earlier that light causes greening of the
cotyledons and thus there was the possibility that this
would alter the results. The plantings were staggered so
that all enzyme assays in a series were done on the same
day. At the end of the required period of germination,
ranging from 0 to 11 days at 2 day intervals, the seedlings
were harvested, the cotyledons were detached the seed coats
removed and the tissue washed with distilled water. Some
cotyledons were used for enzyme assays while others were

10

 

 

11

oven dried at near 100°C for 24 hours and used in the carbo-

hydrate, lipid and protein content determinations.

Preparation of extract for enzyme assays

 

The seven cotyledons used in the enzyme assays were
homogenized in an ice-cold mortar and pestle with 2.5 ml of
0.1M sodium acetate buffer pH 6.0 per cotyledon. The whole
homogenate was used for the a- and B—glycosidase and acid
phosphatase assays. The clear supernatant solution which
resulted when the homogenate was centrifuged at 12,000 rpm
for 30 minutes in a Sorvall Superspeed RC-2 refrigerated
centrifuge, using the SS-34 rotor at about 4°C, was used as
the extract for a—amylase and peroxidase assays.

Five prechilled cotyledons were used in the prepara-
tion of the mitochondria. Malate dehydrogenase activity was
measured by a modification of the method of Nawa and Asahi

(27).

12

Enzyme assays

 

More detailed description of the constituents used
and the conditions employed in the assays and analyses are
given in the Appendix.

The respective para-nitrophenyl-D-glycopyranosides
at the final concentration of 0.01 mg/ml were used as sub-
strates in the assay for the different glycosidases. A
total volume of 1 ml reaction mixture containing substrate,
extract and 0.1M sodium acetate buffer, pH 5.4, was incu-
bated at 35°C for 30 minutes in the case of a- and B-
galactosiase and 90 minutes for the a- and B-glucosidase
assays. At the end of the incubation period, 1.0 ml of
cold 10% (w/v) trichloroacetic acid was added and centri-
fuged at top speed in a clinical centrifuge for about 2
minutes. The supernatant solution was decanted and 2 m1
of 2N NaOH solution added to develop the yellow color of
the liberated nitrOphenol which was read at 405nm in a Beck-
man DB SpectrOphotometer. Appropriate controls were run for
each enzyme assay.

The procedure followed for the assay for acid
phosphatase was as for the glycosidases except that para-

nitrOphenylphosphate, at 0.01 mg/ml final concentration,

13

was the substrate used and the incubation period was five
minutes.

The a-amylase assay was a modification of the method
given by Filner and Varner (11). Extract, starch solution
and 0.1M sodium phosphate buffer, pH 6.9 were incubated at
35°C for a known length of time at the end of which 2.5 ml
of Iodine reagent was added. The blue color which developed
was read at 620nm and the change in the optical density (OD)
was computed. As a control, the extract was added to the
reaction mixture after the Iodine reagent was added.

Peroxidase was determined by the method of Luck (22)
using para-phenylenediamine and hydrogen peroxide as sub-
strates with a pH 6.9 sodium phosphate buffer. The change in
optical density at 485nm was observed for three minutes.

A modification of the method of Nawa and Asahi (27)
was used in the assay for malate dehydrogenase activity.

The change in optical density at 340nm was observed for 5
minutes employing a reaction mixture containing 5mmw sodium
phosphate buffer pH 7.5, lOmM reduced nicotinamide adenine
dinucleotide, lZmM oxalacetic acid, 0.2% (v/v) Triton X-100

and extract.

14

Lipid, protein and carbohydrate analyses

 

The oven dried cotyledons were ground into a fine
powder in a dry mortar and pestle. A known weight of powder
was transfered to a thimble, placed into a Soxhlet apparatus
and extracted for 48 hours with reagent grade chloroform
(10). The chloroform extract was then evaporated to dryness
under reduced pressure in a Buchi Rotavapor in a tared round
bottom flask, weighed, and the percent lipid in the respec-
tive samples computed.

A known weight of the chloroform-extracted powderwas
heated with 2% NaZCO3 in 0.1N NaOH solution in a boiling
water bath for 10 minutes, centrifuged at top speed in the
clinical centrifuge and the protein content of the superna-
tant solution was determined by the Lowry method (21) using
bovine serum albumin as the standard.

The soluble carbohydrate and starch were extracted
from the chloroform-extracted powder by the method of
McCready 32 31. (27). The reducing sugar content was de-
termined using 3,5-dinitrosalicylic acid, with maltose as
the standard (7) whereas the carbohydrate content was de-
termined by the Anthrone method (40). The total carbohy—

drate content was computed as the sum of the soluble carbo-

hydrate and starch values.

15
Respiration measurement

The oxygen uptake and carbon dioxide evolution of
the cotyledons were determined by Warburg manometry at 25°C
(40) in flasks containing 3.0 ml solution and 80 milligrams
of cotyledonary tissue. The tissue was sliced into pieces
of 2 millimeters thickness and the respiratory measurements

were done in duplicate.

Studies with growth regulators

Seeds of the cream/black 'eye' bambarra groundnut
were germinated in vermiculite kept moist with water, 5 ug/
ml gibberellic acid, or 1 ug/ml N6-benzyladenine for either
2 or 10 days. At the end of the germination period, the
cotyledons were harvested, washed with distilled water,
homogenized in 0.1M sodium acetate buffer pH 6.0 and the

assays for the enzymes performed.

RESULTS

Although there is an apparent initial decrease, the
a-amylase activity increases after the first day of germi-
nation in the dark. After the 5th and 9th days of germina-
tion, the rate of increase in the enzyme activity starts to
slow down in the cream/black 'eye' and cream/no 'eye' culti-
vars respectively; however, there is a further sharp increase
in the cream/black 'eye' between teh 9th and llth day (Fig.
l).

Peroxidase activity is virtually absent in the co-
tyledons of the cream/black 'eye' bambarra groundnut during
the early stages of germination, but a significant increase
in activity is observed after the third day (Fig. 2) and
continues to increase further with time. A similar pattern
of development has been reported in Phaseolus mgggg_cotyle-
dons (13).

There is an initial decrease in the a—galactosidase
activity in the cotyledons of the bambarra groundnut seeds
which continues to the 3rd day in the dream/black 'eye' but

for only a day in the cream/no 'eye' cultivar. The rate of

16

Activ1ty (AOD62ohm/cotyledon/minute)

17

2.0‘”

 

n L 1 l
I f I

0 l 3 5 7 9 11

Days germinated

Fig. l.--Changes in a-amylase activity in the cotyledons
of dark-grown bambarra groundnut seeds.
X-—-x cream/no 'eye' O-——O cream/black 'eye'

18

15.0-

10.0-

Activity (A0048Sum/cotyledon/minute)

 

 

Days germinated

Fig. 2.--Peroxidase activity in the cotyledons of dark—
germinated bambarra groundnut seeds (cream/
black 'eye' cultivar).

19

increase which follows, however, tends to slow down with
time in the cream/black 'eye' while it increases in the
cream/no 'eye.’ (Fig. 3). A small but definite increase

in B-galactosidase activity is followed by a subsequent
decrease with time in the two cultivars but the cream/black
'eye' cultivar shows a futher apparent increase on the llth
day of germination (Fig. 4).

Fig. 5 shows the pattern of development of a-
glucosidase in the cream/black 'eye.’ There is an increase
in activity which reaches a maximum on the 5th day of germ-
ination and then declines with time. A similar pattern was
observed in bean hypocotyl tissue (28). The B-glucosidase
activity shows a general pattern of decrease of activity
with time (Fig. 6) which is more evident in the cream/black
'eye' than in the cream/no 'eye' cultivar.

The acid phosphatase development virtually follows
the same pattern in the two bambarra groundnut cultivars.
There is an initial increase followed by a decrease and then
a subsequent further increase with time (Fig. 7).

Fig. 8 shows the development of malate dehydrogenase
activity in the mitochondria and post-mitochondria fractions.
There is a small but significant increase in activity in the

mitochondrialfraction starting early in the germination

Activ1ty (OD4OSnm/cotyledon/minute)

20

.3J

 

Fig. 3.--Changes in a-galactosidase
activity with time in bambarra
groundnut cotyledons grown in
the dark.

X-—-X cream/no 'eye'

O———O cream/black 'eye'

.1-

 

 

l i

o i 5 5 7

w

11

Days germinated

21

 

 

 

1.0 q
(1)
4.)
5
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5 .8+
C.
O
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a <
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4w
0
8
>* .2'
p
-H
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-H
p
0
fl 4 - L
0 I r r I l I
0 1 3 5 7 9 11

Days germinated

Fig. 4.--Changes in the B-galactosidase activity in the
cotyledons of dark-germinated bambarra ground-
nut seeds with time.

X-—-X cream/no 'eye' O-——O cream/black 'eye'

ACthlty (OD405nmx 10/cotyledon/minute)

22

 

 

.20 ‘

.153

.10‘

.051

0 ' I I it* u T
0 l 5 7 9 11

Days germinated

Fig. 5.-—Pattern of a-glucosidase activity in the
cotyledons of cream/black 'eye' cultivar
germinated in the dark.

. . . t .
ACthlty (OD405nmx lO/cotyledon/minu e)

23

.35... Kg 7v:
x/ /\ J

.30“

 

.25”

1A

.20

.15J

.10‘r

 

 

I I I I

I
O 1 3 5 7 9 11

Days germinated

Fig. 6.--Pattern of B-glucosidase activity in the
cotyledons of dark-grown bambarra groundnut
seeds with time.

X-—-X cream/no 'eye' O-——O cream/black 'eye'

Act1v1ty (OD4OSnm/cotyledon/minute)

24

 

 

2.0—
1.5-
2
1.0“
.5“
0 I I I I I T
0 l 3 5 7 9 11

Days germinated

Fig. 7.--Changes in the acid phosphatase activity in the
cotyledons of dark-grown bambarra groundnut
seeds with time.

X-—-X cream/no 'eye' O-——O cream/black 'eye'

25

 

 

.5-

'5

‘3 y

.5 '4‘\
E

\

‘3

c C

a) J

32 .3

p

O

U

\E

3 02"

m

D

O

<

5‘ .1-
.g l
-H

u

U

‘1:

D i I I I l T
0 1 3 5 7 9 11

Days germinated

Fig. 8.--Changes in malate dehydrogenase activity in the
mitochondria and post-mitochondria fractions of
the cotyledons of cream/no 'eye' bambarra
groundnut seed with time.

X-——X total
O--O post-mitochondria
Ar—HA mitochondria

26

process. In the post-mitochondrial fraction, there is an
initial decrease in the enzyme activity till the 5th day
of germination then an increase till the 9th day before a
final decrease. Increased malate dehydrogenase activity
in the mitochondrial fraction of the pea cotyledon during
the first 72 hours of hydration has been reported by Nawa
and Asahi (27). The activity of the enzyme in the post-
mitochondrial fraction remained unchanged in the pea, un-
like what was observed in the bambarra groundnut.

Fig. 9 shows the respiratory pattern of imbibed
cotyledons of the cream/black 'eye' bambarra groundnut.
The oxygen uptake, carbon dioxide evolution and RQ all in-
crease with increased time of imbibition.

The fresh weight of the embryonic axis of the two
cultivars shows an increase after the first day of darke
germination and continues dramatically with age (Fig. 10).
Germination, considered as the protrusion of the radicle
outside the seed coat, is relatively rapid in the two cul-
tivars studied as compared with that reported by Hepper

(15) for 'early germinating' Voandzeia subterranea cv.

 

Subterranea.
The percentage lipid of the cream/black 'eye' cul-

tivar decreases with time (Fig. 11). The values of 10.43

p1 gas/hour

1201

100‘

80‘

60‘

40‘

20-

27

 

 

 

Fig.

I I h I 1
2 3 6 9 21

 

Hours imbibed

9.--Pattern of O uptake, CO evolution and RQ of
the cotyledons of the cream/black 'eye' bambarra
groundnut with length of inhibition.

 

 

1,000" 28 .
800‘
O
600‘
U)
a
(U
i;
.H .
H
H
-H
a
400 “
200‘
/.
o X‘ . . r . . 4
0 1 3 5 7 9 11

Days germinated

Fig. 10.--Changes in the fresh weight of the embryonic
axis of dark-grown bambarra groundnut seeds.

X———X cream/no 'eye,‘ O-——O cream/black 'eye'

29

 

 

15.0%
1
moi
.% M
H
G)
U‘
“3
4.)
C
(D
U
34
CD
D-I
5.0d
0 I r I I I I
O l 3 5 7 9 11

DaysIgerminated

Fig. ll.--Change in percent lipid of the cotyledons of
the dark-germinated cream/black 'eye' bambarra
groundnut seed with time.

30

and 11.56 percent lipid obtained for the dry cotyledons of
the cream/black 'eye' and cream/no 'eye' respectively are
higher than those quoted in the literature (3, 15, 16).

Fig. 12 shows the changes in the percentage protein
of the cotyledons of the dark-grown bambarra groundnut
seeds. The two cultivars show a small overall decrease in
the percentage protein in the cotyledons during dark germi-
nation.

The percentage total carbohydrates (Fig. 13) shows
a general pattern of decrease with time. Fig. 14 shows
the percentage soluble carbohydrates of the cream/no 'eye'
cultivar. There is an increase to the 7th day of dark-
germination and thereafter a decrease. In both cultivars,
there was no appreciable amount of reducing sugar detect-
able in the soluble carbohydrate fraction.

Fig. 15 shows the changes in total carbohydrate;
lipid and protein contents of the cotyledons of the cream/
black 'eye' bambarra groundnut during dark-germination.

The lipid and carbohydrate decrease with time; the protein
content decreased sharply for the first three days and
thereafter the decrease with germination was more gradual.

Table 1 shows the effects of the growth regulators,

BA and GA, on changes of enzyme activities in the cotyledons

30
25
20

c

"-1

w

.p

o

H

m

m

m 15

m

.p

c

m

o

H

S.’
10
5
0

Fig.

31

1

 

 

I | I l I j
0 l 3 5 7 9 11

Days germinated

12.-—Changes in the percent protein of the dark-
grown bambarra groundnut seed cotyledons
with time.
X-——X cream/no 'eye' O-——O cream/black 'eye'

32

 

 

 

50d
1
-(
u 40
a
m
H
m
>
-:-l
5
U‘
0 30“
m
U)
o
o
5
H
o»
m 20-
cu
m
4J
c
m
o
u
m
m
101
0 I I I I I I
0 l 3 5 7 9 11

Days germinated

Fig. l3.--Changes in the percent total carbohydrates of
dark-grown bambarra groundnut seeds with time.
X-——X cream/no 'eye' O-—-O cream/black'eye'

33

 

 

5.0‘
p 4.0
I:
(D
r-I
(U
>
--I
:3
U‘
a) 3.07
(D
U)
0
U
.‘3
H
0"
(D
m 2.0‘
(U
4.)
C
G)
O
H
(D
a;
1.07
Q I 1 I l I I
0 l 3 5 7 9 11

Days germinated

Fig. 14.--Changes in the percent soluble carbohydrates
of dark-germinated cream/no 'eye' bambarra
groundnut with time.

34

milligrams
T350

”300 ‘ .,

-1 250

~200

 

 

 

grams glucose
equivalent

 

 

Days germinated

 

'“100 1.0 "
“50‘ .5 “
I ”T I ’7’ 1

CI 1 3 5 17 11

Fig. lS.--Changes in total carbohydrate, lipid and protein contents with
age of the cotyledons of dark-germinated cream/black 'eye'

bambarra groundnut seeds.
0"". Lipid, O—O Protein,

©—— @ Carbohydrate

35

TABLE l.--The effect of the growth regulators, GA and BA,
on changes in enzymatic activity of dark-
germinated cream/black ‘eye' bambarra groundnut
seed between the 2nd and 10th day. Seeds were
germinated in vermiculate kept moistzwith water,
swg/ml GA solution or lug/ml BA solution.
Changes in activity were computed by subtracting
the values of activity on the 2nd day from that
of the 10th day.

W

Change in Activity1

Ackipmoqiamase aagflactxfidase afimmdase penmddaae

 

 

Control .25 .55 .44 .67
BA 14 ..52 .27 .52
GA .27 .53 53 .88

1

mm phosphatase act1v1ty OD405m/5 min./0.2 ml

a-galactosidase activity = OD2105 m/30 min /0 2 ml
aeamylase actIV1ty = A0062: /4:m1n./50u1

Perox1dase act1v1ty = Aw485m/3 mun/50111

36

of dark-germinated cream/black 'eye' bambarra groundnut
seeds between the 2nd and 10th days of growth. The changes
in activity were computed by subtracting the values of ac—
tivity on the 2nd day from that of the 10th day. BA in-
hibited the development of increases in activities of acid
phosphatase, a-amylase and peroxidase but had no effect on
the activity of a-galactosidase. GA also had no effeCt on
acid phosphatase and a—galactosidase. However, the GA
treatment resulted in increased activity of a-amylase and
peroxidase.

The BA and GA treatments had no effects on the ac-
tivities of a- and B-glucosidase (data not given) in the
cotyledons of dark-germinated cream/black 'eye' bambarra

groundnut seeds.

DISCUSSION

The findings reported here are far from being com-
plete since only a small fraction of the overall metabolism
of the bambarra groundnut was investigated. The changes
observed, however, are not too different from what has been
observed during germination of seeds of some related species.

The major findings of this research are: l) A dra-
matic increase in peroxidase, from virtually no activity
in the dry seed, with germination. 2) Increase during ger-
mination, after an initial decrease, in a-amylase and a-

galactosidase activities, CO evolution, and R0. 3) An

2
initial increase followed by decrease with germination of
the activities of B-galactosidase, a-glucosidase and mito-
chondria fraction malate dehydrogenase. 4) Decrease in
B—glucosidase activity, lipid, carbohydrate and protein
content during germination. 5) An initial increase fol-
lowed by a decrease before the final increase in acid

phosphatase activity and 0 uptake with germination. 6)

2

An initial decrease followed by an increase and then fin-

ally decrease in the activity of the post-mitoChondria

37

38

malate dehydrogenase. 7) GA treatment resulted in the in-
creased activity of a-amylase, and peroxidase, while the
BA treatment inhibited the increase in activity of acid
phosphatase, a-amylase and peroxidase, Both the GA and

BA had no effects on a—galactosidase, a- and B-glucosidase
activities. The GA also had no effect on acid phosphatase
activity.

The dry seeds of the bambarra groundnut had some
activity of all the enzymes studied, except peroxidase.
This probably is residual activity of enzymes present in
the seeds during development. These different enzymes in
the cotyledons, however, showed different patterns of de-
velOpment during dark-germination.

The increase in the a-amylase activity in the bam-
barra groundnut is similar to that reported for the mung
bean, peas, and barley although the activity is detected
about five days earlier in the bambarra than in the dark-
grown peas (38).

The increase in the a-amylase is as expected (in
order to degrade the starch present in the bambarra ground-
nut seed) but the decrease in the a-glucosidase activity

with age is contrary to what is expected.

39

The dramatic increase in the a-galactosidase sug-
gests that the bambarra groundnut seeds contain appreciable
amounts of galactans. The decrease in the B—glucosidase,
however, is reasonable since few beta glucans act as food
reserve. However, since no attempts were made to charac-
terize the sugars present in the bambarra groundnut seeds,
nothing can be said about this at present.

The increase in peroxidase activity is the largest
noted but since the exact function of this enzyme has not
been established, the significance of this increase in the
bambarra groundnut can not be assessed.

Seeds usually store inorganic phosphates required
during germination in the form of inositol hexaphosphate
or phytic acid and the early increase of phytase activity
provides the phosphate for the growing embryonic axis.

But no appreciable phytase activity was detected in the
germinating bambarra groundnut seeds. Thus the form of
phosphate storage, if any, in the bambarra groundnut seed
is not known. Because of the non-specific nature of the
substrate used, the acid phosphatase activity noted may be
due to a composite of many enzymes.

The change in malate dehydrogenase activity of the

mitochondria fraction is similar to that observed in the

40

peas (27). This is probably an indication of the changes
in the mitochondria themselves as observed in the peanut
(4). The decrease in the post-mitochondrial malate dehy4
drogenase activity corresponds with the increase in the
activity in the mitochondria fraction (Fig. 8). The fur-
ther decrease in the activity of this enzyme may be due
to disintegration of the enzyme with time.

The increase in both CO2 evolution and 02 uptake
may be because as the time of imbibition increases, more
of the organelles involved in the respiratory function of

cotyledons are stimulated to action. The initial decrease

in CO evolution, RQ and increase in O

2 uptake may be due

2
to the cotyledons adjusting to the injury caused them
through slicing. The low RQ is similar to that reported
for some seeds during the early stages of germination (24).
The fact that the cream/no 'eye' cultivar has been
subjected to much more rigorous selection practices than
the cream/black 'eye' cultivar probably accounts for the
higher protein in the dry seeds of the former than the
latter. The lipid and total carbohydrate contents of the

two cultivars are very similar and like the protein con-

tent, decrease with germination. The decrease in protein

41

content with time during germination is similar to that
reported for peanuts (4).

BA treatment has been reported to cause a delay in
cotyledon senescence in the pea (36). This is probably the
result of an inhibition of increase in activity of the en-
zymes which hydrolyze the stored materials. The delayed
increase in a—amylase activity in the BA treated bambarra
groundnut cotyledons is similar to that reported on the pea
(17, 37). Acid phosphatase, a-amylase and peroxidase,
whose activities were delayed by the BA treatment are prob-
ably associated with the cotyledon senescence process. GA
treatment has the opposite effect on these enzymes, except
acid phosphatase. The increase in peroxidase activity in
the GA treated bambarra groundnut cotyledons is similar to
the observation of Michniewicz and Stanislawski (26).

The effects of the growth regulators, gibberellic
acid and benzyladenine, on the levels of some enzymes indi-
cate that endogenous growth chemicals probably can affect
the metabolism of the bambarra groundnut.

Finally, the results of these exploratory, prelimin-
ary studies showed that although the embryonic axes of both
cultivars developed at about the same rate, the percentage

protein in the cream/no 'eye' was about twice that of the

42

cream/black 'eye.‘ The cream/no 'eye' had lower levels of
a-amylase, a-galactosidase and acid phosphatase activities
and these tended to develop somewhat more slowly than in
the other cultivar.

Thus it is hoped that the observations and changes
documented here will serve as a basis for other more com-
plete and thorough research on bambarra groundnut. Al-
though a staple in the diet in certain regions of Africa,
it is not used to any appreciable extent in the West. Per-
haps further exploration will reveal its nutritional poten-
tial. It is also possible that certain unique characteris-
tics which it possesses will make it desirable for scien-

tific studies.

APPENDIX

APPENDIX

Grinding medium for preparation of the

 

mitochondrial and post-mitochondrial fractions

 

lOmM potassium phosphate buffer pH 7.2

0.7M mannitol (12.75 gm-100 ml)

1 . OmM ethylene diamine tetraacetic acid (380 mg/100 ml)

0.1% (w/v) Bovine serum albumin

0.05% (w/v) cysteine

Grind 5 prechilled cotyledons with 20 ml. of the

grinding medium. Filter through 4 layers of cheesecloth
and centrifuge at 270 x g for 5 minutes. Suspend the pre-
cipitate in 10 ml of above medium minus cysteine and cen-
trifuge for another 5 minutes. Centrifuge the supernatant
solution at 25,300 x g for 30 minutes. Resuspend the pre-
cipitate in a known volume of the medium minus cysteine.
This is the mitochondria fraction. The supernatant solu-
tion of the 25,300 x g certrifugation is the post-

mitochondria fraction.

43

44

Assay for malate dehydrogenase activity

Total of 3.0 ml containing:
2.0 ml lSOmM potassium phosphate buffer pH 7.5
0.1 ml NADH (2 mg/ml solution)
0.2 ml Oxalacetic acid (4 mg/ml solution)
0.5 ml 0.2% Triton X-100 (1.2 ml/10 ml)
0.2 ml extract
Read opitcal density at 340nm at zero time and after 5

minutes.

Assay for a—amylase activity

Starch substrate. Heat in a boiling water bath 2.5

 

mg/ml starch suspension for 1—1/2 minutes. The suspending

medium is a solution of 40mM KH PO containing 0.2 mM CaCl

2 4 2'

Cool and centrifuge at 17,300 x g for 30 minutes and use

the supernatant solution as substrate.

Iodine reagent. Six percent KI, 0.6% I2 in water.

Dilute loo-fold with 0.05N HCL and use as Iodine reagent.

 

45
Assay_procedure

0.1 ml of extract

0.2 ml of 2.5 mg/ml starch substrate

0.7 ml 0.1M solution phosphate buffer pH 6.9
Incubate for a known period of time. Add 2.5 m1

of Iodine reagent and reat at 620nm.

Assay of peroxidase activity

Total of 3.0 ml assay mixture containing:
2.5 ml 0.1M sodium phosphate buffer pH 6.9
0.2 m1 of 0.1% para-phenylenediamine
0.2 ml of H202 solution (prepared by diluting

0.33 ml 30% H202 to 100 ml)
0.1 m1 extract.

Read the optical density at zero time and after

3 minutes.

Protein determination by the Lowry method

To 0.8 ml of solution add 5.0 ml of Reagent C (pre-

pare daily by mixing 50 ml of 2% Na2CO3 in 0.1N NaOH with

46

1 ml of 0.5% cupric sulphate in 1% tartrate). Allow to
stand for exactly 10 minutes. Add 0.8 ml of Folin or
Phenol reagent. Mix thoroughly and allow to stand for

30 minutes before reading at 660nm.

Anthrone method for carbohydrates

 

Reagent. Dissolve 2.0 gm anthrone in a litre of

95% H 80 (prepared by adding a litre of concentrated HZSO

2 4 4

cautiously to 50 ml water with cooling).

Procedure. To 3 ml of sample, add 6 ml of reagent.

 

Mix thoroughly at once. Place in boiling water bath for 4

minutes. Cool and read at 620nm.

Reducing sugars determination

Color reagent. Dissolve 1 gm of 3,5-dinitrosali-

 

cylic acid in 20 ml of 2N NaOH (8 gm/100 ml). Add 30 gm
sodium-potassium tartrate to 50 ml water and warm to dis-
solve. Mix the two solutions and make up to 100 ml.

Procedure. To 1 ml of sugar solution add 1 m1 of

 

color reagent. Boil for 5 minutes in a boiling water bath.

Cool and add 2 ml of water and read at 540nm.

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Plant Physiol. 35:288

 

VER ITY LIBRARIES

1 III I

7

MICHIGAN STAT

 

I II III II
3 1293 0316‘!