Theoretical and Applied G e n e t i c s 46, 19-23 (1975) 9 by S p r i n g e r - V e r l a g 1975
Differences of Cytoplasmic Transaminase Activity in Normal and Opaque-2 Maize (Zea mays L.) Seedlings" I. Langer, P . Str~nsk~ and M. Kut~cek P l a n t B r e e d i n g Station, Stupice, and Czechoslovak A c a d e m y of Sciences, Institute of E x p e r i m e n t a l Botany, P r a g u e (Czechoslovakia) Summary. The activity of glutamate-oxalacetate and glutamate-pyruvate transaminases (GOT 2.6. I. I., GPT 2.6.1.2. ) was determined in seedlings of normal and opaque-2 maize. The activity of these transaminases was determined in homozygotes +/+ and o2/os, their F i hybrid, in mixture of +/+ and +/o~ in S~F 4 generation. The GOT and GPT activity in mutants opaque-2 was always higher than in normal plants. In generation S~F 4 the higher activity of transaminases in op~ue-2 plants was demonstrated without addition of the pyridoxal-5-phosphate (PRP) coenzyme (at GOT I, 36x, at GPT 2,00x) as well as at the optimum PRP concentration (at GOT 1,39x, at GPT 2, 48x). The difference in transaminase activity is therefore not due to changes in PRP biosynthesis. The determination of the GOT and GPT activity in seedlings might be used in breeding experiments as a screening method for selection of mutants.
Jones
and Singleton,
in the late 20 's, described
the mu-
tant gene
op~ue-2, which results in the typical opaque
structure
of the maize
nothing was
known
kernel
endosperm.
of the effect of this mutant
the nutritive value of the maize (1964)
made
recessive
a break-through
op~-2
gene
sential amino
However,
endosperm. when
nearly
gene on Mertz
et al.
they found that the
doubles
the limiting es-
acids content in the endosperm
of maize
kernel. Interconversien a basic cowise terested
of amino
of amino
in whether
acids by transamination
acid metabolism
the alteration of amino
sis by the op~ue-2 gene is manifest aminase
is
; we were
in-
acid synthe-
in a modified
trans-
recessive mutant op~-2 gene, kindly given by Prof. A. Bianchi**. Their SoF• hybrid, and plants grown from phenotypically normal kernels of segregating ears of the S~F~ and S~F 3 generations, were also examined. The transaminase activity inthe generation S~F 4 was determined in normal homozygous plants (from non-segregating ears- genotype +/+), in mutant homozygous plants (from segregated op~-2 kernels - genotype 02/02 ) and in plants from phenotypically normal kernels of a segregating ear - genotypes +/+ and 2/02 (see Fig. I). For parents +/+ and 02/02 , and in the subsequent generations up to SmFj, the activity was determined by means of an average sample taken out of 30 seedlings. In the $3F4 generation the activity was determined by four replications of I0 seedlings each. The seeds were sterilized for 2 minutes in 10 % formaldehyde, rinsed and left for 6 hours in distilled water before sowing. The seeds were placed on damp cellulose and cultivated at 23 ~ C in the dark.
activity.
In the present
investigation,
the glutamate-oxalacetate
transaminase
and of the glutamate-pyruvate I. 2. ) was
followed
and op~ue-2 maize,
the enzymatic
in seven
(GOT
transaminase day-old
with the aim
seedlings
activity of 2.6.1.1. (GPT
2.6.
of normal
of finding out whether
or not the transaminase
activity of the two genotypes
ready
at an early ontogenic
shows
Materials
differences
)
al-
phase.
Preparation of the e n z y m e extract
Seven days after sowing the shoots were cut, weighed and cooled down to 4 ~ C. Cool (4 ~ C) phosphate buffer 0.1M at pH 8, containing 0, 4M saccharose, 0,01M EDTA and 0,01M 2-mercaptoethanol (Wightman and Cohen 1968) was used for extraction. The plant material together with the buffer (I : 3) was ground in the mixer at 4~ The mixture was left for 30 minutes in solid CO 2 cooled bath, then filtered through linen cloth and centrifuged for 20 minutes at 4 ~ C and 12.000 g. Supernatant (enzyme extract, EE) was separated and used for estimating the enzymatic activity.
and Methods
Plant material
and its cultivation
Determination of the e n z y m e activity
Transaminase activity was determined on the inbredline SW 437 (normal endosperm) of a collection from the Plant Breeding Station, Stupice, and in the S~ line derived from the synthetic population homozygous for the
The G O T and G P T activity w a s determined by a modified m e t h o d of C a b a u d et al. (1956), originally used for the determination of transaminase activity in blood s e r u m . The catalyzed enzymatic reactions have this course:
* Dedicated to Prof. Borriss on the occasion of his 65th
** Istituto Sperimentale per la Cerealicoltura, R o m a ,
anniversary
Italia.
20
I. Langer
et al. : Cytoplasmic
Transaminase
Opaque-2 Maize
Activity in
120 p.m
Seedlings
0 n
100
ih 6o
III
!i'ti,'
..,. ..,- oZl o /o 20
/'
o
i
/
/
a 0
-'+
2'
.z
/
I
06
a
8
7 pH
9
Z5
30
35 40 ~ 45 Temlerature
0
600 I
n --e
0
F i g . 1. P h e n o t y p e a n d g e n o t y p e r e p r e s e n t a t i o n of Zea mays e v o l v e d in S t u p i c e . 1 S p e c i f i c a t i o n , 2 G e n e r a t i o n , 3 P h e n o t y p e of e n d o s p e r m , 4 G e n o t y p e of s e e d l i n g
40O
3
300
0
2OO
0
/ 4J ~
L-aspartate + ~-ketoglutarate ....
L-alanin
L-glutamate + pyruvate
W e determined the concentrations of the resulting ketoacids, i.e. oxaloacetate in G O T and pyruvate in G P T . B y adding anilincitrate reagent, oxaloacetate in the first case w a s converted to pyruvate. Pyruvate w a s chemically stabilized in the form of hydrazone by using 2, 4-dinitrophenylhydrazine. Hydrazone w a s shaken into toluene and determined at 490 n m on a U n i c a m SP 1800 spectrophotometer. The o p t i m u m values for pH, temperature, incubation period and concentration of the c o e n z y m e pyridoxal - 5 phosphate ( P R P ) are given in Fig. 2. In our w o r k w e u s e d p H = 8, 380 C, 50 ~g P R P per ml and 20 minutes incubation period. T w o series of e n z y m e activity determination w e r e investigated simultaneously in three different genotypes (+/+, +/o2 , o2/o2 ) of the generation S~F 4 , with and without addition of the c o e n z y m e (50 ~g/ml). The resulting activity of G O T and G P T is expressed in international enzymologic units for I m g protein (I.U./mg).
o f protein
in enzyme
The proteins in EE were determined (1951). Human serum albumin was
100 0
c
25 50 75 .,.g/m[125 pffp-----,,-
ses, the GOT
and GPT
the phenotypically from GPT
mutant
SISyn
activity tended
mained.
after Lowry et al. used as standard.
vity 2 times zygotes
o 2 was
not differ in protein ties were
different
content
activities
1,8
times
higher,
mal
SW
nance
(GPT)
were
1,4
and GPT
showed
form
times
respectively,
F 1 hybrid
of the low GOT
observed
and GPT
(Tab. I). In the mutant
the transaminase
437. The
but the GOT
F1,
did activi-
S 1 Syno2,
(GOT)
and
to decrease
+/+,
30 rain40
additional higher
than in 2790/2
form
GPT
higher)
PRP
o 2 re-
the activity
and the GPT
+ (mixture +/o2).
system
with
is about the same
(GOT
acti-
of homo-
From
the ana-
transaminase
but the relation between
and
genera-
+ and
we found that these differences
in the enzyme
and mutant 2,48x
in successive
times
from
of the GOT
the mutants
and of heterozygotes
(50 ~g/ml),
grown
lower than in plants
values
at 0, 1 level. The whole
vity is higher PRP
1,36
higher
were
The absolute
In the S3F 4 without
of 2790/2
02, and their hybrid
20 lime
of seedlings
seeds
tions but the relation between
significant 437,
activities
normal
seeds.
lysis of variance
The lines SW
10
Fig. 2. Determination of optimum conditions for measuring GOT (full line) and GPT (dotted line) transaminase activity in maize ( ~M ofpyruvic acid -PyA- evolved) a Optimum pH, b Optimum temperature, c Effect of coenzyme PRP concentration, d Time course of transaminase reaction
extracts
Results
/
//
L-glutamate + oxaloacetate
+ ~-ketoglutarate
Determination
/ !
addition
were actiof
the normal 1,39 x higher,
(Tab. 2 and 3).
than in the nor-
a complete
activities.
in the SIF 2 and S2F 3 generations.
The
domisame
was
In all ca-
Discussion The most likely way in which the amino acid composition of the endosperm proteins is shifted by a structural
I. Langer
Table
et al. : Cytoplasmic
1. Protein
Year
Transaminase
content and GOT
Specification
Activity in 0paque-2
and GPT
Generation
activity in seven
Embryo geno-
Endosperm
type
pheno-
Maize
days
Seedlings
old seedlings
Proteins mg/ml
GOT U.I./
21
(without coenzyme
GPT U.I./
addition)
Proteins % *
GOT % *
GPT % *
m g proteiri
EE
type
1968
SiSyno 2
P
0202
02
5,79
1,30
0,72
103,3
144,4
180,0
SW 437
P
++
+
5,61
0,90
0,40.
100,0
100,0
100,0
1969
S i S y n o 2 x S W 437
S0F 1
+o 2
+
5,88
0,95
0,36
99,5
105,4
90,0
1970
366
SIF 2
++, +o 2
+
5,55
0,85
0,41
99,0
94,4
102,5
1971
1155
S2F 3
++, +02
+
5,52
0,87
0,38
98,4
96,6
95,0
1972
2790/2
S3F 4
++, +o 2
+
5,56
0,75
0,25
99,2
83,4
62,5
2790/2
S3F 4
o2o 2
o2
5,73
1,10
0,54
102,2
122,1
135,0
2790
S3F 4
++
+
5,53
0,80
0,23
98,5
88,9
57,5
* sw
437 = 100J
Table 2. P r o t e i n content and GOT and GPT activity in s e v e n days old s e e d l i n g s of the SsF 4 g e n e r a t i o n Specification
Genotype
Proteins mg/ml EE
• 102
Activity GOT U.I./mgprot. without coenzyme
Activity GPT U.I./mg prot. •
• 102
with addition
without coenzyme
with addition
2790/5
++
561,4
a*
89,4
a
277,8
a
39,4
a
86,8
a
2790/2
++, +02
557,6
a
94,8
a
292,2
a
35,6
a
88,2
a
2790/2
o2o 2
560,0
a
129,0
b
399,0
b
71,0
b
205,8
b
* Values marked with the same letter do not differ significantly; ficantly on the probability level P = 0,01 gene mutation
is by a change
single protein fractions.
opaq~-2 amine
synthesis
albumine
of the nutritionally unsuitable
prol-
of nutritionally
The changes,
for in the regulating suppose be played
to be partly suppressed,
and globuline
son 1970).
valuable
fractions
systems
proteins
of protein synthesis. role might
changed
conditions
with different letters differ signi-
and s t a l k s c a u s e d by the gene 02. They pointed out that the gene opaque-2 b e g i n s to function at the m i l k y - w a x stage. Murphy and Dalby (1971) i n v e s t i g a t i n g the p r o tein f r a c t i o n s and Mehta et al. (1972) following DNA and RNA
(Nel-
milar
opinion that gene
mation
of the kernels.
(1973)
reported
We
additionally the amino
of proteosynthesis.
op~ue-2
marked
in the
have to be looked
by the ability to quickly rearrange
W h e r e a s the effect of the
where-
is de-repressed
therefore,
that a very important
acid pool under
of the gene
fraction seems
as the synthesis
in the proportions
In the case of the mutant
values
gene on the c o m -
fractions
and RNA-ase
in the germ
cent comparative
study of free and bound and
tail, our knowledge about the effect of this gene on the
of two varieties:
the free amino
e n z y m e s y s t e m of a m i n o acid m e t a b o l i s m and about pro-
siderably
in opaque-2
teosynthesis in the vegetative plants is less complete.
acids content did not differ much
So far there is no consensus as to w h e n gene o 2 starts
The differences
to influence plant metabolism. Alexander et al. (1969)
GOT,
observed no difference in the a m i n o acid composition
found in our present
of the proteins of the e m b r y o and the leaf of a n o r m a l
these results.
and mutant plant. ~ev6enko and Agafonov (1972) w e r e
single enzymes
not able to detect differences in R N A
for maize,
content in leaves
and normal
may
Mar~lek
change 1972).
acid
plants we acid levels
acids content was
plants,
the bound
con-
amino
(Str~nsk~r et al. ,1974). activity,
seven
investigation
It is, however,
amino
in free amino
in transaminase
in opaq~.e-2
and
In our re-
ten day-old
found significant differences
higher
composi-
maize.
content in normal
position of the e n d o s p e r m protein is k n o w n in m o r e de-
et al.
in the embryo
and normal
opctq~-P
the for-
Kadam
in the isozyme
dehydrogenase
of opaque-~
to a si-
o 2 is acting only during On the other hand,
differences
tion of the glutamate
activity came
seedlings
are in agreement
known during
especially
day-old
with
that the activity of ontogenesis
The question
(e.g.
still remains
I. Langer et al . : C y t o p l a s m i c T r a n s a m i n a s e A c t i v i t y in Opaque-2 M a i z e S e e d l i n g s
22
Table
3. Variance analysis generation
Source of variability
of protein
Proteins mg/ml EE
(d.F.)
content
and GOT,
Activity GOT without coenzyme
• 102
F
MS
GPT
MS
U.I./mg prot. with addition F
+
2
18,45
12
3,87
Genotype Residue
2 . 3 0 5 , 8 61,9
whether
the changes
tivity observed
in young
plants persist
the later
acids
C 4 connected
related
the photosynthetic causing
levels.
to the Krebs
an increase
with our results
with the Krebs
is apparent.
We
tion of amino
cycle,
assume
while
on transaminases
an inverted
accumula-
with the Krebs
with the enhanced
cycle
no difference
is
activity of their in-
and mutant
for primarily
maize.
From
therefore,
of amino
and GPT
the GOT
the coenzyme
not act through
acids
PRP
show
that the gene
biosynthesis;
of practical
amino
the differen-
breeding
opaque-2
are not
such
contemporary
work
yield,
slower
(Alexander
forms
and without
as lower
cold resistance,
it is es-
mutant
acid composition
side-effects,
lowered
transami-
of the coenzyme.
the standpoint
with an improved
et al. 1969, show
opaque
fractions,
it is, however,
and GPT Methods
methods
(Kamra
test based
activity in young
conditions. (e.g.
be possible normal
•
1972).
to grow
out for clinical human
amino
they
after selecting
op-
plant
allow the select-
In this way
the segregating
opaque-2 cross seedlings
with changed
Calbiochem,
to our experience
it would
to maturity.
to identify among
On
be sugge-
only a part of the young
first leaf),
their
on the determination
plants may
well worked
Taking
For
activity determination
and others) ; according
for analysis
ker-
to adopt suitable,
can also be applied to plant material timum
normal
(see the test sets of the firms
B6hringer
some
et al. 1971;
but with a high proportion
of transaminase
and very
Already
can be obtained.
necessary
and rapid screening a screening
genes. Pollaczek
that phenotypically
structure,
protein
our results, of GOT
37,8
those
it would progeny
of
individuals
acid metabolism.
related
and GPT
activity in both genotypes
by addition
the undesirable
etc. From
of valuable
simple
++"~
22.515,2 595,6
activity should belooked
sential to be able to select the
endosperm,
transaminase
suppose,
40,2
F
Literature
of pursuing
with and without
cancelled
was
cycle.
results
ces in GOT
We
in transaminase in the metabolism
to the Krebs
o 2 does
we found that there
in the activity of tryptophan
that differences
nases
investigation
1.888,4
of modifying
Paez
et al. 1973)
ed indivudual
In a separate
The
(e.g.
biochemistry
dependence
MS +
by a number
results
are simple
terconversion.
in normal
fluenced
sted.
and glycine
that the decreased
acids connected
again connected
of
C 3 simultaneously
in the serine
F
10 S
F001 = 12,97
selection
the photosynthe-
cycle decreases
MS
47,0
nels without
with the formation
activity of type
Compared
connected
between
of photosynthesis.
opaque-2 mutant
found that in the
rises,
differences
plants in the incorporation
into the first metabolites
tic activity of type amino
studied
opaque-2 maize
and
14CO2
They
ac-
of the S3F 4
U.I./mg prot. with addition
+++
2 1 . 4 0 4 , 4 355,5
Peter
Jolivet et al. (1970)
of
and GPT
during
Activity GPT without coenzyme
F
F01 = 6,93
in GOT
old seedlings
62,2
of ontogenesis.
normal
MS
37,2
to be answered
days
102
+++
4,77
F05 = 2,88
phases
activity at seven
soft
ripening
et al. 1969,
1971; S a l a m i n i et al. 1970; Chadzinov 1971) it can be c o n c l u d e d that the final effect of opo.que-2 gene is in-
A l e x a n d e r , D . E . , Dudley, J . W . , L a m b e r t , R . J . : The m o d i f i c a t i o n of p r o t e i n quality of m a i z e by b r e e d i n g . In: E u c a r p i a , A k a d e m i a i Kiado, Bu d ap est , 33-43 (1971) A l e x a n d e r , D . E . , L a m b e r t , R . J . , Dudley, J . W . : B r e e d ing p r o b l e m s and p o t e n t i a l s of modified p r o t e i n m a i z e . In: New a p p r o a c h e s to b r e e d i n g for i m p r o v e d plant p r o t e i n , IAEA Vienna, 55-56 (1969) Cabaud, P . , L e e p e r , R . , W r b b l e w s k i , F . : C o l o r i m e t r i c m e a s u r e m e n t in s e r u m : g l u t a m a t e - o x a l a c e t a t e , g l u t a m a t e - p y r u v a t e t r a n s a m i n a s e . A m e r . J . Clin. P at h . 26, 1101-1105 (1956) Chad~inov, M . I . : D i r e c t i o n and methods of b r e e d i n g f o r i m p r o v i n g p r o t e i n , l y s i n e and o t h e r aci ds c o n tent in g r a i n . In: S e l e k c i j a i s e m e n o v o d s t v o k u k u r u z y , Kolos, Moskva, 17-32 (1971) J o l i v e t , E . , Nicol, M . , Baudet, J . , M o s s e , Y . : Diff 6 r e n c e s dans 1 ' i n c o r p o r a t i o n du 14CO 2 ~ la l u m i ~ r e c h e z de j e u n e s m a i z e n o r m a l et opo.q~-2. In: I m -
I. Langer et a l . : C y t o p l a s m i c T r a n s a m i n a s e A c t i v i t y in Opaque-2 M a i z e S e e d l i n g s
23
p r o v i n g plant p r o t e i n by n u c l e a r t e c h n i q u e s . IAEA Vienna ( 1970 ) K a d a m , S . S . , Singh, J . , Mehta, S.L. : C h a n g e s in i s o e n z y m e s in e m b r y o and opaque-2 Zea mays during inhibition. P h y t o c h e m . 12, 1221-1225 (1973) K a m r a , O . F . : B r e e d i n g and s c r e e n i n g t e c h n i q u e s f o r s e e d p r o t e i n i m p r o v e m e n t in c r o p p l a n t s . In: Induced m u t a t i o n s and plant i m p r o v e m e n t , IAEA, V i e n na, 303-315 (1972) Lowry, H . O . R o s e n b r o u g h , N . J . , F a r r , A . L . , Randall, R . J . : P r o t e i n m e a s u r e m e n t with the F o l i n Phenol R e a g e n t . J . B i o l . C h e m . 193, 265-275 (1951 Mar~s L. : E n z y m e a c t i v i t y of Zea mays genotype in r e l a t i o n to the h e t e r o s e e f f e c t . Genetika a ~ l e c h t~nf 8, 109-114 (1972) Mehta, ~ . L . , S r i v a s t a v a , K . N . , Mall, P . C . , Naik, M . S . : Changes in the n u c l e i c acid and p r o t e i n f r a c tions in opaque-2 m a i z e k e r n e l s during d e v e l o p m e n t . P h y t o c h e m . 1...~1, 937-942 (1972) M e r t z , E . , B a t e s , L . , N e l s o n , O. : Mutant gene that c h a n g e s c o m p o s i t i o n and i n c r e a s e s l y s i n e content of m a i z e e n d o s p e r m . S c i e n c e 145, 279-280 (1964) Murphy, 3 . J . , Dalby, A. : C h a n g e s in the p r o t e i n f r a c tion of developing n o r m a l and opaque-2 m a i z e en d o s p e r m . C e r e a l C h e m . 4._88, 336-348 (1971) N e l s o n , O . E . : I m p r o v i n g of plant p r o t e i n quality. In: p r o v i n g plant p r o t e i n by n u c l e a r t e c h n i q u e s , IAEA Vienna, 43-51 (1970)
P a e z , A o V , , H e l m , J . L . , Z u b e r , M.So : Lysine content of opaque-2 m a i z e k e r n e l s having d i f f e r e n t phenotypes C r o p Scio 9, 251-252 (1969) Pes~ev, N . , Je-leni~, D . , ~ukalovi~, V. : S o m e ge ne t i c and b i o c h e m i c a l c h a r a c t e r i s t i c s of a new s o u r c e of m a i z e opaque-2 mutant. T h e o r e t . Appl. G e n e t i c s 4..~3, 23-26 (1973) P o l l a c z e k , M . , C a u d e r o n , A . , Caen en , M. : K e r n e l t e x t u r e and l y s i n e content in opaque-2 m a i z e . In: E u c a r p i a , A k ad ~ m i ai Kiadb, Bu d ap est , 49-53 (1971) S a l a m i n i , F . , B o r g h i , B . , L o r e n z o n i , C. : The effect of the opaque-2 gene on y i e l d in m a i z e . E u p h y t i c a 19, 531-538 (1970) ~ev~enko, V . E . , Agafonov, N . S . : D e t e r m i n a t i o n of the p h y s i o l o g i c a l - b i o c h e m i c a l action of the opaque-2 gene at the h o m o z y g o t h y b r i d s of m a i z e . V~stnik s e l s k o c h o z j a j s t v e n n o j nauki No. 1, 55-57 (1972) Str~nsk~, P . , Kut~t~ek, M . , Lan g er , I . , J i r ~ e k , V ~ Iqbal, J . : C o m p a r i s o n of the effect of g a m a r a d i a tion on the am i n o acid content and t r a n s a m i n a s e a c tivity of n o r m a l and opaque-2 m a i z e d i f f e r i n g i n t h e i r n i t r o g e n m e t a b o l i s m . Rad i at i o n Botany, 1974 Wightman, F . , Cohen, D. : I n t e r m e d i a r y s t e p s in the e n z y m a t i c c o n v e r s i o n of tryptophan to IAA in c e l l f r e e s y s t e m s f r o m M u n g bean s e e d l i n g s . In" W i g h t man, F . and S e t t e r f i e l d , G. ( E d . ) , B i o c h e m i s t r y and P h y s i o l o g y of Plant Growth S u b s t a n c e s , pp. 275286~ Ottawa: Runge P r e s s 1968
R e c e i v e d O c t o b e r 15, 1974
D r . I. Langer Plant B r e e d i n g Station 25084 Stupice ( C z e c h o s l o v a k i a )
C o m m u n i c a t e d by H. Stubbe
D r . P . Str&nsk~ D o z . D r . M. Kut&6&k C z e c h o s l o v a k A c a d e m y of S c i e n c e s Institute of E x p e r i m e n t a l Botany Ke dvoru 16/15 16630 P r a h a 6 - V o k o v i c e ( C z e c h o s l o v a k i a )
Differences of Cytoplasmic Transaminase Activity in Normal and Opaque-2 Maize (Zea mays L.) Seedlings" I. Langer, P . Str~nsk~ and M. Kut~cek P l a n t B r e e d i n g Station, Stupice, and Czechoslovak A c a d e m y of Sciences, Institute of E x p e r i m e n t a l Botany, P r a g u e (Czechoslovakia) Summary. The activity of glutamate-oxalacetate and glutamate-pyruvate transaminases (GOT 2.6. I. I., GPT 2.6.1.2. ) was determined in seedlings of normal and opaque-2 maize. The activity of these transaminases was determined in homozygotes +/+ and o2/os, their F i hybrid, in mixture of +/+ and +/o~ in S~F 4 generation. The GOT and GPT activity in mutants opaque-2 was always higher than in normal plants. In generation S~F 4 the higher activity of transaminases in op~ue-2 plants was demonstrated without addition of the pyridoxal-5-phosphate (PRP) coenzyme (at GOT I, 36x, at GPT 2,00x) as well as at the optimum PRP concentration (at GOT 1,39x, at GPT 2, 48x). The difference in transaminase activity is therefore not due to changes in PRP biosynthesis. The determination of the GOT and GPT activity in seedlings might be used in breeding experiments as a screening method for selection of mutants.
Jones
and Singleton,
in the late 20 's, described
the mu-
tant gene
op~ue-2, which results in the typical opaque
structure
of the maize
nothing was
known
kernel
endosperm.
of the effect of this mutant
the nutritive value of the maize (1964)
made
recessive
a break-through
op~-2
gene
sential amino
However,
endosperm. when
nearly
gene on Mertz
et al.
they found that the
doubles
the limiting es-
acids content in the endosperm
of maize
kernel. Interconversien a basic cowise terested
of amino
of amino
in whether
acids by transamination
acid metabolism
the alteration of amino
sis by the op~ue-2 gene is manifest aminase
is
; we were
in-
acid synthe-
in a modified
trans-
recessive mutant op~-2 gene, kindly given by Prof. A. Bianchi**. Their SoF• hybrid, and plants grown from phenotypically normal kernels of segregating ears of the S~F~ and S~F 3 generations, were also examined. The transaminase activity inthe generation S~F 4 was determined in normal homozygous plants (from non-segregating ears- genotype +/+), in mutant homozygous plants (from segregated op~-2 kernels - genotype 02/02 ) and in plants from phenotypically normal kernels of a segregating ear - genotypes +/+ and 2/02 (see Fig. I). For parents +/+ and 02/02 , and in the subsequent generations up to SmFj, the activity was determined by means of an average sample taken out of 30 seedlings. In the $3F4 generation the activity was determined by four replications of I0 seedlings each. The seeds were sterilized for 2 minutes in 10 % formaldehyde, rinsed and left for 6 hours in distilled water before sowing. The seeds were placed on damp cellulose and cultivated at 23 ~ C in the dark.
activity.
In the present
investigation,
the glutamate-oxalacetate
transaminase
and of the glutamate-pyruvate I. 2. ) was
followed
and op~ue-2 maize,
the enzymatic
in seven
(GOT
transaminase day-old
with the aim
seedlings
activity of 2.6.1.1. (GPT
2.6.
of normal
of finding out whether
or not the transaminase
activity of the two genotypes
ready
at an early ontogenic
shows
Materials
differences
)
al-
phase.
Preparation of the e n z y m e extract
Seven days after sowing the shoots were cut, weighed and cooled down to 4 ~ C. Cool (4 ~ C) phosphate buffer 0.1M at pH 8, containing 0, 4M saccharose, 0,01M EDTA and 0,01M 2-mercaptoethanol (Wightman and Cohen 1968) was used for extraction. The plant material together with the buffer (I : 3) was ground in the mixer at 4~ The mixture was left for 30 minutes in solid CO 2 cooled bath, then filtered through linen cloth and centrifuged for 20 minutes at 4 ~ C and 12.000 g. Supernatant (enzyme extract, EE) was separated and used for estimating the enzymatic activity.
and Methods
Plant material
and its cultivation
Determination of the e n z y m e activity
Transaminase activity was determined on the inbredline SW 437 (normal endosperm) of a collection from the Plant Breeding Station, Stupice, and in the S~ line derived from the synthetic population homozygous for the
The G O T and G P T activity w a s determined by a modified m e t h o d of C a b a u d et al. (1956), originally used for the determination of transaminase activity in blood s e r u m . The catalyzed enzymatic reactions have this course:
* Dedicated to Prof. Borriss on the occasion of his 65th
** Istituto Sperimentale per la Cerealicoltura, R o m a ,
anniversary
Italia.
20
I. Langer
et al. : Cytoplasmic
Transaminase
Opaque-2 Maize
Activity in
120 p.m
Seedlings
0 n
100
ih 6o
III
!i'ti,'
..,. ..,- oZl o /o 20
/'
o
i
/
/
a 0
-'+
2'
.z
/
I
06
a
8
7 pH
9
Z5
30
35 40 ~ 45 Temlerature
0
600 I
n --e
0
F i g . 1. P h e n o t y p e a n d g e n o t y p e r e p r e s e n t a t i o n of Zea mays e v o l v e d in S t u p i c e . 1 S p e c i f i c a t i o n , 2 G e n e r a t i o n , 3 P h e n o t y p e of e n d o s p e r m , 4 G e n o t y p e of s e e d l i n g
40O
3
300
0
2OO
0
/ 4J ~
L-aspartate + ~-ketoglutarate ....
L-alanin
L-glutamate + pyruvate
W e determined the concentrations of the resulting ketoacids, i.e. oxaloacetate in G O T and pyruvate in G P T . B y adding anilincitrate reagent, oxaloacetate in the first case w a s converted to pyruvate. Pyruvate w a s chemically stabilized in the form of hydrazone by using 2, 4-dinitrophenylhydrazine. Hydrazone w a s shaken into toluene and determined at 490 n m on a U n i c a m SP 1800 spectrophotometer. The o p t i m u m values for pH, temperature, incubation period and concentration of the c o e n z y m e pyridoxal - 5 phosphate ( P R P ) are given in Fig. 2. In our w o r k w e u s e d p H = 8, 380 C, 50 ~g P R P per ml and 20 minutes incubation period. T w o series of e n z y m e activity determination w e r e investigated simultaneously in three different genotypes (+/+, +/o2 , o2/o2 ) of the generation S~F 4 , with and without addition of the c o e n z y m e (50 ~g/ml). The resulting activity of G O T and G P T is expressed in international enzymologic units for I m g protein (I.U./mg).
o f protein
in enzyme
The proteins in EE were determined (1951). Human serum albumin was
100 0
c
25 50 75 .,.g/m[125 pffp-----,,-
ses, the GOT
and GPT
the phenotypically from GPT
mutant
SISyn
activity tended
mained.
after Lowry et al. used as standard.
vity 2 times zygotes
o 2 was
not differ in protein ties were
different
content
activities
1,8
times
higher,
mal
SW
nance
(GPT)
were
1,4
and GPT
showed
form
times
respectively,
F 1 hybrid
of the low GOT
observed
and GPT
(Tab. I). In the mutant
the transaminase
437. The
but the GOT
F1,
did activi-
S 1 Syno2,
(GOT)
and
to decrease
+/+,
30 rain40
additional higher
than in 2790/2
form
GPT
higher)
PRP
o 2 re-
the activity
and the GPT
+ (mixture +/o2).
system
with
is about the same
(GOT
acti-
of homo-
From
the ana-
transaminase
but the relation between
and
genera-
+ and
we found that these differences
in the enzyme
and mutant 2,48x
in successive
times
from
of the GOT
the mutants
and of heterozygotes
(50 ~g/ml),
grown
lower than in plants
values
at 0, 1 level. The whole
vity is higher PRP
1,36
higher
were
The absolute
In the S3F 4 without
of 2790/2
02, and their hybrid
20 lime
of seedlings
seeds
tions but the relation between
significant 437,
activities
normal
seeds.
lysis of variance
The lines SW
10
Fig. 2. Determination of optimum conditions for measuring GOT (full line) and GPT (dotted line) transaminase activity in maize ( ~M ofpyruvic acid -PyA- evolved) a Optimum pH, b Optimum temperature, c Effect of coenzyme PRP concentration, d Time course of transaminase reaction
extracts
Results
/
//
L-glutamate + oxaloacetate
+ ~-ketoglutarate
Determination
/ !
addition
were actiof
the normal 1,39 x higher,
(Tab. 2 and 3).
than in the nor-
a complete
activities.
in the SIF 2 and S2F 3 generations.
The
domisame
was
In all ca-
Discussion The most likely way in which the amino acid composition of the endosperm proteins is shifted by a structural
I. Langer
Table
et al. : Cytoplasmic
1. Protein
Year
Transaminase
content and GOT
Specification
Activity in 0paque-2
and GPT
Generation
activity in seven
Embryo geno-
Endosperm
type
pheno-
Maize
days
Seedlings
old seedlings
Proteins mg/ml
GOT U.I./
21
(without coenzyme
GPT U.I./
addition)
Proteins % *
GOT % *
GPT % *
m g proteiri
EE
type
1968
SiSyno 2
P
0202
02
5,79
1,30
0,72
103,3
144,4
180,0
SW 437
P
++
+
5,61
0,90
0,40.
100,0
100,0
100,0
1969
S i S y n o 2 x S W 437
S0F 1
+o 2
+
5,88
0,95
0,36
99,5
105,4
90,0
1970
366
SIF 2
++, +o 2
+
5,55
0,85
0,41
99,0
94,4
102,5
1971
1155
S2F 3
++, +02
+
5,52
0,87
0,38
98,4
96,6
95,0
1972
2790/2
S3F 4
++, +o 2
+
5,56
0,75
0,25
99,2
83,4
62,5
2790/2
S3F 4
o2o 2
o2
5,73
1,10
0,54
102,2
122,1
135,0
2790
S3F 4
++
+
5,53
0,80
0,23
98,5
88,9
57,5
* sw
437 = 100J
Table 2. P r o t e i n content and GOT and GPT activity in s e v e n days old s e e d l i n g s of the SsF 4 g e n e r a t i o n Specification
Genotype
Proteins mg/ml EE
• 102
Activity GOT U.I./mgprot. without coenzyme
Activity GPT U.I./mg prot. •
• 102
with addition
without coenzyme
with addition
2790/5
++
561,4
a*
89,4
a
277,8
a
39,4
a
86,8
a
2790/2
++, +02
557,6
a
94,8
a
292,2
a
35,6
a
88,2
a
2790/2
o2o 2
560,0
a
129,0
b
399,0
b
71,0
b
205,8
b
* Values marked with the same letter do not differ significantly; ficantly on the probability level P = 0,01 gene mutation
is by a change
single protein fractions.
opaq~-2 amine
synthesis
albumine
of the nutritionally unsuitable
prol-
of nutritionally
The changes,
for in the regulating suppose be played
to be partly suppressed,
and globuline
son 1970).
valuable
fractions
systems
proteins
of protein synthesis. role might
changed
conditions
with different letters differ signi-
and s t a l k s c a u s e d by the gene 02. They pointed out that the gene opaque-2 b e g i n s to function at the m i l k y - w a x stage. Murphy and Dalby (1971) i n v e s t i g a t i n g the p r o tein f r a c t i o n s and Mehta et al. (1972) following DNA and RNA
(Nel-
milar
opinion that gene
mation
of the kernels.
(1973)
reported
We
additionally the amino
of proteosynthesis.
op~ue-2
marked
in the
have to be looked
by the ability to quickly rearrange
W h e r e a s the effect of the
where-
is de-repressed
therefore,
that a very important
acid pool under
of the gene
fraction seems
as the synthesis
in the proportions
In the case of the mutant
values
gene on the c o m -
fractions
and RNA-ase
in the germ
cent comparative
study of free and bound and
tail, our knowledge about the effect of this gene on the
of two varieties:
the free amino
e n z y m e s y s t e m of a m i n o acid m e t a b o l i s m and about pro-
siderably
in opaque-2
teosynthesis in the vegetative plants is less complete.
acids content did not differ much
So far there is no consensus as to w h e n gene o 2 starts
The differences
to influence plant metabolism. Alexander et al. (1969)
GOT,
observed no difference in the a m i n o acid composition
found in our present
of the proteins of the e m b r y o and the leaf of a n o r m a l
these results.
and mutant plant. ~ev6enko and Agafonov (1972) w e r e
single enzymes
not able to detect differences in R N A
for maize,
content in leaves
and normal
may
Mar~lek
change 1972).
acid
plants we acid levels
acids content was
plants,
the bound
con-
amino
(Str~nsk~r et al. ,1974). activity,
seven
investigation
It is, however,
amino
in free amino
in transaminase
in opaq~.e-2
and
In our re-
ten day-old
found significant differences
higher
composi-
maize.
content in normal
position of the e n d o s p e r m protein is k n o w n in m o r e de-
et al.
in the embryo
and normal
opctq~-P
the for-
Kadam
in the isozyme
dehydrogenase
of opaque-~
to a si-
o 2 is acting only during On the other hand,
differences
tion of the glutamate
activity came
seedlings
are in agreement
known during
especially
day-old
with
that the activity of ontogenesis
The question
(e.g.
still remains
I. Langer et al . : C y t o p l a s m i c T r a n s a m i n a s e A c t i v i t y in Opaque-2 M a i z e S e e d l i n g s
22
Table
3. Variance analysis generation
Source of variability
of protein
Proteins mg/ml EE
(d.F.)
content
and GOT,
Activity GOT without coenzyme
• 102
F
MS
GPT
MS
U.I./mg prot. with addition F
+
2
18,45
12
3,87
Genotype Residue
2 . 3 0 5 , 8 61,9
whether
the changes
tivity observed
in young
plants persist
the later
acids
C 4 connected
related
the photosynthetic causing
levels.
to the Krebs
an increase
with our results
with the Krebs
is apparent.
We
tion of amino
cycle,
assume
while
on transaminases
an inverted
accumula-
with the Krebs
with the enhanced
cycle
no difference
is
activity of their in-
and mutant
for primarily
maize.
From
therefore,
of amino
and GPT
the GOT
the coenzyme
not act through
acids
PRP
show
that the gene
biosynthesis;
of practical
amino
the differen-
breeding
opaque-2
are not
such
contemporary
work
yield,
slower
(Alexander
forms
and without
as lower
cold resistance,
it is es-
mutant
acid composition
side-effects,
lowered
transami-
of the coenzyme.
the standpoint
with an improved
et al. 1969, show
opaque
fractions,
it is, however,
and GPT Methods
methods
(Kamra
test based
activity in young
conditions. (e.g.
be possible normal
•
1972).
to grow
out for clinical human
amino
they
after selecting
op-
plant
allow the select-
In this way
the segregating
opaque-2 cross seedlings
with changed
Calbiochem,
to our experience
it would
to maturity.
to identify among
On
be sugge-
only a part of the young
first leaf),
their
on the determination
plants may
well worked
Taking
For
activity determination
and others) ; according
for analysis
ker-
to adopt suitable,
can also be applied to plant material timum
normal
(see the test sets of the firms
B6hringer
some
et al. 1971;
but with a high proportion
of transaminase
and very
Already
can be obtained.
necessary
and rapid screening a screening
genes. Pollaczek
that phenotypically
structure,
protein
our results, of GOT
37,8
those
it would progeny
of
individuals
acid metabolism.
related
and GPT
activity in both genotypes
by addition
the undesirable
etc. From
of valuable
simple
++"~
22.515,2 595,6
activity should belooked
sential to be able to select the
endosperm,
transaminase
suppose,
40,2
F
Literature
of pursuing
with and without
cancelled
was
cycle.
results
ces in GOT
We
in transaminase in the metabolism
to the Krebs
o 2 does
we found that there
in the activity of tryptophan
that differences
nases
investigation
1.888,4
of modifying
Paez
et al. 1973)
ed indivudual
In a separate
The
(e.g.
biochemistry
dependence
MS +
by a number
results
are simple
terconversion.
in normal
fluenced
sted.
and glycine
that the decreased
acids connected
again connected
of
C 3 simultaneously
in the serine
F
10 S
F001 = 12,97
selection
the photosynthe-
cycle decreases
MS
47,0
nels without
with the formation
activity of type
Compared
connected
between
of photosynthesis.
opaque-2 mutant
found that in the
rises,
differences
plants in the incorporation
into the first metabolites
tic activity of type amino
studied
opaque-2 maize
and
14CO2
They
ac-
of the S3F 4
U.I./mg prot. with addition
+++
2 1 . 4 0 4 , 4 355,5
Peter
Jolivet et al. (1970)
of
and GPT
during
Activity GPT without coenzyme
F
F01 = 6,93
in GOT
old seedlings
62,2
of ontogenesis.
normal
MS
37,2
to be answered
days
102
+++
4,77
F05 = 2,88
phases
activity at seven
soft
ripening
et al. 1969,
1971; S a l a m i n i et al. 1970; Chadzinov 1971) it can be c o n c l u d e d that the final effect of opo.que-2 gene is in-
A l e x a n d e r , D . E . , Dudley, J . W . , L a m b e r t , R . J . : The m o d i f i c a t i o n of p r o t e i n quality of m a i z e by b r e e d i n g . In: E u c a r p i a , A k a d e m i a i Kiado, Bu d ap est , 33-43 (1971) A l e x a n d e r , D . E . , L a m b e r t , R . J . , Dudley, J . W . : B r e e d ing p r o b l e m s and p o t e n t i a l s of modified p r o t e i n m a i z e . In: New a p p r o a c h e s to b r e e d i n g for i m p r o v e d plant p r o t e i n , IAEA Vienna, 55-56 (1969) Cabaud, P . , L e e p e r , R . , W r b b l e w s k i , F . : C o l o r i m e t r i c m e a s u r e m e n t in s e r u m : g l u t a m a t e - o x a l a c e t a t e , g l u t a m a t e - p y r u v a t e t r a n s a m i n a s e . A m e r . J . Clin. P at h . 26, 1101-1105 (1956) Chad~inov, M . I . : D i r e c t i o n and methods of b r e e d i n g f o r i m p r o v i n g p r o t e i n , l y s i n e and o t h e r aci ds c o n tent in g r a i n . In: S e l e k c i j a i s e m e n o v o d s t v o k u k u r u z y , Kolos, Moskva, 17-32 (1971) J o l i v e t , E . , Nicol, M . , Baudet, J . , M o s s e , Y . : Diff 6 r e n c e s dans 1 ' i n c o r p o r a t i o n du 14CO 2 ~ la l u m i ~ r e c h e z de j e u n e s m a i z e n o r m a l et opo.q~-2. In: I m -
I. Langer et a l . : C y t o p l a s m i c T r a n s a m i n a s e A c t i v i t y in Opaque-2 M a i z e S e e d l i n g s
23
p r o v i n g plant p r o t e i n by n u c l e a r t e c h n i q u e s . IAEA Vienna ( 1970 ) K a d a m , S . S . , Singh, J . , Mehta, S.L. : C h a n g e s in i s o e n z y m e s in e m b r y o and opaque-2 Zea mays during inhibition. P h y t o c h e m . 12, 1221-1225 (1973) K a m r a , O . F . : B r e e d i n g and s c r e e n i n g t e c h n i q u e s f o r s e e d p r o t e i n i m p r o v e m e n t in c r o p p l a n t s . In: Induced m u t a t i o n s and plant i m p r o v e m e n t , IAEA, V i e n na, 303-315 (1972) Lowry, H . O . R o s e n b r o u g h , N . J . , F a r r , A . L . , Randall, R . J . : P r o t e i n m e a s u r e m e n t with the F o l i n Phenol R e a g e n t . J . B i o l . C h e m . 193, 265-275 (1951 Mar~s L. : E n z y m e a c t i v i t y of Zea mays genotype in r e l a t i o n to the h e t e r o s e e f f e c t . Genetika a ~ l e c h t~nf 8, 109-114 (1972) Mehta, ~ . L . , S r i v a s t a v a , K . N . , Mall, P . C . , Naik, M . S . : Changes in the n u c l e i c acid and p r o t e i n f r a c tions in opaque-2 m a i z e k e r n e l s during d e v e l o p m e n t . P h y t o c h e m . 1...~1, 937-942 (1972) M e r t z , E . , B a t e s , L . , N e l s o n , O. : Mutant gene that c h a n g e s c o m p o s i t i o n and i n c r e a s e s l y s i n e content of m a i z e e n d o s p e r m . S c i e n c e 145, 279-280 (1964) Murphy, 3 . J . , Dalby, A. : C h a n g e s in the p r o t e i n f r a c tion of developing n o r m a l and opaque-2 m a i z e en d o s p e r m . C e r e a l C h e m . 4._88, 336-348 (1971) N e l s o n , O . E . : I m p r o v i n g of plant p r o t e i n quality. In: p r o v i n g plant p r o t e i n by n u c l e a r t e c h n i q u e s , IAEA Vienna, 43-51 (1970)
P a e z , A o V , , H e l m , J . L . , Z u b e r , M.So : Lysine content of opaque-2 m a i z e k e r n e l s having d i f f e r e n t phenotypes C r o p Scio 9, 251-252 (1969) Pes~ev, N . , Je-leni~, D . , ~ukalovi~, V. : S o m e ge ne t i c and b i o c h e m i c a l c h a r a c t e r i s t i c s of a new s o u r c e of m a i z e opaque-2 mutant. T h e o r e t . Appl. G e n e t i c s 4..~3, 23-26 (1973) P o l l a c z e k , M . , C a u d e r o n , A . , Caen en , M. : K e r n e l t e x t u r e and l y s i n e content in opaque-2 m a i z e . In: E u c a r p i a , A k ad ~ m i ai Kiadb, Bu d ap est , 49-53 (1971) S a l a m i n i , F . , B o r g h i , B . , L o r e n z o n i , C. : The effect of the opaque-2 gene on y i e l d in m a i z e . E u p h y t i c a 19, 531-538 (1970) ~ev~enko, V . E . , Agafonov, N . S . : D e t e r m i n a t i o n of the p h y s i o l o g i c a l - b i o c h e m i c a l action of the opaque-2 gene at the h o m o z y g o t h y b r i d s of m a i z e . V~stnik s e l s k o c h o z j a j s t v e n n o j nauki No. 1, 55-57 (1972) Str~nsk~, P . , Kut~t~ek, M . , Lan g er , I . , J i r ~ e k , V ~ Iqbal, J . : C o m p a r i s o n of the effect of g a m a r a d i a tion on the am i n o acid content and t r a n s a m i n a s e a c tivity of n o r m a l and opaque-2 m a i z e d i f f e r i n g i n t h e i r n i t r o g e n m e t a b o l i s m . Rad i at i o n Botany, 1974 Wightman, F . , Cohen, D. : I n t e r m e d i a r y s t e p s in the e n z y m a t i c c o n v e r s i o n of tryptophan to IAA in c e l l f r e e s y s t e m s f r o m M u n g bean s e e d l i n g s . In" W i g h t man, F . and S e t t e r f i e l d , G. ( E d . ) , B i o c h e m i s t r y and P h y s i o l o g y of Plant Growth S u b s t a n c e s , pp. 275286~ Ottawa: Runge P r e s s 1968
R e c e i v e d O c t o b e r 15, 1974
D r . I. Langer Plant B r e e d i n g Station 25084 Stupice ( C z e c h o s l o v a k i a )
C o m m u n i c a t e d by H. Stubbe
D r . P . Str&nsk~ D o z . D r . M. Kut&6&k C z e c h o s l o v a k A c a d e m y of S c i e n c e s Institute of E x p e r i m e n t a l Botany Ke dvoru 16/15 16630 P r a h a 6 - V o k o v i c e ( C z e c h o s l o v a k i a )
