Download DISTRIBUTION OF GROWTH AND ENZYME ACTIVITY IN THE

DISTRIBUTION OF GROWTH AND ENZYME ACTIVITY IN THE
DEVELOPING GRAIN OF WHEAT
By A. H. G. C. RIJVEN* and R. COHEN*
[Manuscript received April 14, 1961]
Summary
Ears of field-grown wheat were harvested on 12 occasions between anthesis
and ripeness. Fresh weight, dry weight, and total nitrogen content of selected
grains were determined.
On eight occasions similar grains were dissected into two future grain coat
parts, designated "outer pericarp" and "rest of grain coat", and into endosperm
and embryo. The resulting four fractions were analysed for total and for soluble
protein content, for chlorophyll content, and for activity of glutamyl transferase,
acid phosphatase, alanine-glutamic acid transaminase, and to a limited extent
of alcohol aehydrogenase.
The results emphasize that the different parts of the grain successively
commence their growth cycles and show distinct biochemical differentiation.
Glutamyl transferase activity is almost exclusively found in future grain
coat tissues. A peak of activity, shown by the whole grain at day 12, can be
accounted for by a peak of activity in the outer pericarp at that day. Specific
activity is highest in this part of the grain.
Transaminase activity is very high in the endosperm even on a protein basis.
The embryo shows the highest specific activity for alcohol dehydrogenase.
Acid phosphatase is found in all tissues but its specific activity is highest
in those that show an early breakdown. In the outer pericarp acid phosphatase
activity remains high after day 12 whilst glutamyl transferase activity and protein
content drop.
The rest of the grain coat has similar enzyme activities to other parts but
it is unique in its high chlorophyll content.
The mechanism establishing the distribution of glut amyl transferase is
discussed.
1. INTRODUCTION
The implications of structure and biochemical differentiation for an understanding of the development of the wheat grain were brought to the fore in this
Laboratory when it was found (Rijven and Banbury 1960) (i) that the enzyme
glutamyl transferase showed a peak of activity early in the development of the
grain; (ii) that this enzyme was almost exclusively localized in the maternal group
of tissues; and (iii) that these tissues gradually degenerated after an early period
of vigorous growth.
Dramatic changes occur in the grain between anthesis and ripeness. Such
changes have been recognized in histological descriptions (Kudelka 1875; Percival
1921; Krauss 1933; Nutman 1939), but their significance has not yet been elucidated
in physiological and biochemical terms.
* Division of Plant Industry,
C.S.I.R.O., Canberra.
WHEAT GRAIN DEVELOPMENT
553
This paper presents a more detailed quantitative study of the development
of the grain and its various parts in terms of protein content and enzyme activity.
Thus not only growth was measured, but also biochemical differentiation.
II.
MATERIAL AND METHODS
(a) Plant Culture and Sampling
In 1960, wheat (Triticum 8ativum L. cv. Festival) was grown in the field in
rows 18 in. apart.
After emergence of the ears, those showing anthesis were tagged daily. On
November 8 the day's yield of ears showing anthesis for the first time was 465. These
ears were immediately retagged at random with labels of 12 different colours. This
device enabled quick harvesting of a random sample on a particular harvest day.
On such a day, 37 ears tagged with the same colour were collected. Harvests were
made for the first seven times at 4-day intervals, then three times at 6-day intervals,
and finally twice at 8-day intervals.
Out of each ear two grains were taken, viz. those of the first floret from two
spikelets in the middle of the ear, i.e. one from each side of the ear. Thirty-six
grains (one from each pair) were dissected and analysed as detailed below. Thirty
of the remaining grains were used for determinations of fresh weight, dry weight,
and total nitrogen content. The remaining grains were fixed in alcohol-formol-acetic
acid for histological studies. The ears were stored deep-frozen.
(b) Di88ection and De8ignation of Part8
Before and after dissection, the 36 grains were kept cool. Dissection was
done for the first time on day 8 and was aimed throughout at separation of parts
differing in cytological origin and metabolic function. For histological orientation
see Plate 1.
(i) The Maternal Group of Ti88ue8.-This group of tissues, i.e. the future grain
coat, comprises the pericarp, integuments, and nucellus. It was divided into two
fractions: "outer pericarp" and "rest of the grain coat".
The outer pericarp is parenchymatous and colourless, except near the groove
where it contains some chlorophyll. Its separation is possible throughout due to
early schizogeny of tissue bordering on the outside of the chlorophyll layer, and is
achieved by a dismantling operation starting from the abaxial side of the grain.
Near the groove, cutting with a fine scalpel is necessary to effect separation.
The rest of the grain coat is rather heterogeneous. Dissection of this fraction
was started from the groove. Its main living part is the chlorophyll layer, which
belongs to the inner layer of the pericarp. Another bulky component is the vascular
bundle with adherent nucellus in the groove.
On the harvest of day 42 the rest of the grain coat and the endosperm were
taken together.
(ii) End08perm8.-These could be easily isolated until day 16 inclusive, but
the operation was continued until day 36 inclusive, although it became increasingly
554
A. H. G. C. RIJVEN AND R. COHEN
more difficult, due to adhesion of the future aleurone layer to the membranous
nucellus, which in fact had to be made part of the endosperm fraction. The number
of endosperms and of grain coats sampled was reduced to 18 from day 24 onwards.
This allowed replacement of any faulty dissections and made grinding, quantitative
transfer, sampling, and extraction easier. Nevertheless, on the last two occasions,
the endosperm and rest of the grain coat fractions may have been contaminated
with each other. The results indicate that the general picture cannot have been
seriously affected by this concession to practicability.
(iii) Embryos.-Embryos constituted a separate fraction for the first time
on day 16, but were, at this stage, only a minute part (see Table 2).
(c) Preparation of Extracts
Each fraction, representing the equivalent of either 36 or 18 grains, was ground
in a chilled mortar in the presence of 0 . 005M tris buffer [tris(hydroxymethyl)aminomethane], pH 7 '4, then transferred quantitatively and made up to a volume of 9 ml.
Samples to a total of 3 ml were withdrawn for determination of chlorophyll and
total protein nitrogen content.
The extracts were then centrifuged for 10 min at 500 g and 1°C, the supernatants
collected, and the residues resuspended in 6 ml of distilled water. The suspensions
were again centrifuged. The combined supernatants were, if necessary, made up to
12 ml, each ml now representing 2 or 1 grain equivalents, and centrifuged for 60 min
at 100,000 g and 1°C.
The supernatants, after this high-speed centrifugation, were used for several
enzyme assays, and for protein nitrogen determinations. In the case of transaminase,
two volumes of saturated ammonium sulphate were added to an aliquot of the
supernatant. This was then centrifuged at 5000 g for 30 min and precipitates were
redissolved in 0 . 10M phosphate buffer, pH 7 ·4.
(d)
Estimation.~
and Enzyme Assay8
(i) Fre8h and Dry Weights.-Fresh weights and dry weights (after drying overnight at 80°C) were determined on six replicates of five grains. For the harvest
taken on the day of anthesis, however, these determinations were made on five
replicates of 10 grains.
(ii) Nitrogen Determinations.-These were carried out on the dried grains
and on the samples drawn from extracts. These samples were treated with
trichloroacetic acid to a final concentration of 5% and the precipitates subjected
to a microKjeldahl digestion procedure followed by nesslerization of the distillates,
using Vanselow reagent. The estimates of samples, drawn before low- and after
high-speed centrifugation, were taken to represent, respectively, total and soluble
proteint content. Whatever the exact interpretation of the latter fraction may be,
it forms the logical basis for calculation of specific activities as the enzyme as~ys
were made on these supernatants.
(iii) Chlorophyll.-Chlorophyll was estimated by adding 4 volumes of acetone
to 1 volume of crude extract and, after centrifugation, reading the optical density
at 652 m/-, (Arnon 1949).
555
WHEAT GRAIN DEVELOPMENT
(iv) Glutamyl Transferase.-The procedure is essentially similar to the one used
by Stumpf, Loomis, and Michelson (1951); 15-ml centrifuge tubes containing 0·1 ml
O·OlM MnS0 4 , 0·1 ml O·OOlM adenosine triphosphate, 0·1 ml 2M NH 20H.HOI
adjusted with NaOH to pH 6·5, 0·5 ml 0·2M tris-maleate buffer, pH 6·5, 0·2 ml
O·2M sodium arsenate, pH 6·5, 0·5 ml 0·5M glutamine extract (i.e. high-speed
supernatant), and water to 2·25 ml were incubated at 30°0 for lO min and longer.
The reaction was stopped by adding 0·75 ml of a mixture consisting of equal parts
of lO% FeOls in 0 ·IN HOI, 50% conc. HOI, and 24% trichloroacetic acid (Oowgill
and Pardee 1957). After centrifugation, the optical density was measured at 540 mfL.
Incubation mixtures without added glutamine were used as blanks. On no occasion
did the optical density of these exceed a value of 0·020. Incubations, for periods
TABLE
1
WEIGHT AND TOTAL NITROGEN DATA OF DEVELOPING GRAIN OF WHEAT
Days
after
Anthesis
0
4
8
12
16
20
24
30
36
42
50
liS
Fresh
Weight
(mg)
2·65
10·93
19·98
27·67
40·17
49·18
57·95
65·08
71·79
74·12
65·83
50·63
Dry
Weight
(mg)
0·69
2·77
4·86
7·92
11·94
15·99
21·63
29·04
37·21
42·27
45·40
43·59
Water
Content
(mg)
1·96
8·16
15·12
19·75
28·23
33·19
36·32
36·04
34·58
31·85
20·43
7·04
Solids
(%)
26·03
25·34
24·32
28·62
29·72
32·51
37·33
44·62
51·83
57·03
68·96
86·10
Total
Nitrogen
(",g)
31·3
77·2
135·7
213·5
305·0
388·6
496·0
681·8
878·0
1126·6
1137·0
up to 20 min, showed that the activity of extracts (O.D.'s <0 ·30) was linear with time,
but with endosperm and embryo extracts, which had to be incubated for periods up
to 1 hr, strict linearity was not always obtained because of very low activity
(O.D.'s <0 ·lO).
(v) Acid Phosphatase.-The assay followed a procedure described by Axelrod
(1947). I-ml aliquots of extract were incubated with 5 ml of 0·277M disodium
p-nitrophenol phosphate in Mj30 acetate buffer, pH 4·8, at 30°0. At regular time
intervals 0·2-ml samples were withdrawn and added to 5 ml Nj6 NaOH and the
optical density measured at 400 mfL. Readings obtained from samples taken after 5
and 25 min of incubation were used to establish activity because it tended to decrease
after longer periods of incubation.
(vi) Transaminase.-The assay followed a procedure described by Oohen
(1955). Ammonium sulphate precipitates (66% saturation) redissolved in 0 ·IM
phosphate buffer, pH 7 ·4, were used as the material to be assayed. 0·5-ml aliquots
556
A. H. G. C. RIJVEN AND R. COHEN
of the purified extract were incubated at 30°C for several time intervals up to 1 hr
with 100 fLmoles alanine, 100 fLmoles a-ketoglutarate, and with 0 ·IM phosphate buffer,
pH 7 '4, to a total volume of2·2 ml. Pyridoxal phosphate was not added (Cruickshank
and Isherwood 1958). The glutamic acid formed was determined manometrically
using bacterial glutamic acid decarboxylase (Worthington Biochem. Corp., U.S.A.).
On occasions, the results showed some unsystematic deviations from linearity with
time, but the effect of error on the assessment of activity was reduced by taking all
individual readings into account.
eo
70
60
50
~
.!
~ 40
I!I
iii
~
30
20
10
o
60
DAYS AFTER ANTHESIS
Fig. I.-Fresh and dry weights of whole grain.
(vii) Alcohol Dehydrogenase.-The assay followed a procedure described by
Racker (19';5). Into a quartz cell (1 cm light path) was pipetted: 0·1 ml 0·06M
diphosphopyridine nucleotide, 0·5 ml 0 ·IM sodium pyrophosphate, pH 8· 5, 0·1 ml
3M ethanol, 2·2 ml water, and extract up to 0·1 ml. The optical density at 340 mfL
was measured at I-min intervals at a constant room temperature of 19°C.
III.
RESULTS
(a) Growth Pattern of Whole Grain
Table 1 summarizes data on fresh weight, dry weight, and total nitrogen
content of whole grains and includes derived data on water content and percentage
solids .. The weight data are plotted in Figure 1, in which it is seen that there were
557
WHEAT GRAIN DEVELOPMENT
no obvious deviations from the familiar sigmoid curve. This is noteworthy because
the weather had shown considerable variation.
(b) Distribution of Protein in Grain Parts
For convenience of comparison, all primary data on grain parts have been
accumulated into a single table (Table 2).
Direct information on grain parts was obtained for the first time on day 8.
The main contributor to the total protein at this harvest is the outer pericarp (see
also Fig. 2). There is no increase of protein in this fraction after this stage. However,
(a)
(b)
800
~
3- 600
0-75
Z
w
C)
~
I-
Z
o
~ 0-50
400
Z
'"
W
~
0..
200
o
10
20
30
40
10
20
30
40
DAYS AFTER ANTHESIS
Fig. 2.-Distribution of protein nitrogen content in grain parts. (a) Additive plot in absolute
values. (b) Additive plot in ratios of the whole grain. 0, outer pericarp; R, "rest of coat"; E,
endosperm; G, embryo (= germ).
it can be inferred from the data on the whole grain of day 0 and 4 that it must have
started from a much lower value, and contributed then probably more to the total
protein of the grain than all other parts taken together. A decrease in protein content,
which continues, is noticed for the first time on day 16. The soluble protein fraction
of the outer pericarp also follows this pattern.
A rise and fall in the total and soluble protein fraction is also shown by the
rest of the grain coat. However, the increase continues until day 20 and higher
absolute values are attained.
The importance of the maternal group of tissues during early development
can be judged from the fact that until day 16 they contribute more than half of the
total protein content of the grain (Fig. 2).
In contrast to these future grain coat tissues, the endosperm and embryo show a
continuing increase in total protein content. The endosperm eventually contains
close to 90% of that of the whole grain. It may be noted that less than 20% of the
endosperm protein is soluble.
558
A. H. G. C. RIJVEN AND R. COHEN
TABLE
2
DISTRIBUTION OF GROWTH AND ENZYME ACTIVITY IN THE DEVELOPING GRAIN OF WHEAT
All data on a grain basis: W = whole grain, 0 = outer pericarp, R = "rest of coat", E = endo·
sperm, G. = germ
-~-~~rain-~~tal
after
Anthesis
_ _ ~_
Part
(/Lg)
W
4
W
8
W
----
Nitrogen
Chloro-I
phyll
Content
(/Lg)
(/Lg)
(/Lg)
~lutam~l-··
Transferase
77·2
37·7
14·9
130·7
69·8
28·2
39·3
22·8
7·7
15·1
9·6
3·5
106·2
43·3
I 4·59
39·3
39·9
27·0
16·5
16·1
10·7
71·3
213·5
o
R
E
305·0
0·98
I
o
34·7
53·7
95·5
G
1·6
W
388·6
2·09
3·18
0·338
2·715
0·450
0·014
0·264
0·069
0·005
,
R
E
G
4·8
W
496·0
o
R
E
G
30
---------
4·648
0·612
7·59
0·38
4·06
0·15
3·750
0·870
0·028
0·350
0·215
0·047
0·39
2·69
4·51
7·08
3·192
0·956
37·40
I 2·160
0·387
0·356
0·213
0·74
4·41
32·25
I
I
12·0
20·1
39·2
0·990
0·042
------------~-----I--~-
o
-~
0·213
-----
183·9
R
E
---20
aminase
(/Lmoles/hr)
(~O.D./hr) (~O.D./hr)
---~------------
W
--I~;:ans--
-----------1--------------
E
16
Acid
Phosphatase
[_0_.1_5_ _ ~~_ _ _ ~03_6_1 ____ _
19· 9
R
W
Soluble
Protein
Nitrogen
__
31_._3_
o
12
Total
Protein
Nitrogen
W
681·8
o
R
E
G
I
269·9
90·8
2·852
1·093
45·61
28·3
56·6
185·0
II ·8
19·8
59·2
1 ·605
1·170
0·077
0·398
0·350
0·345
0·32
1·78
43·51
401·9
118·4
1·161
48·11
23·5
50·8
316·6
H·O
7·0
16·7
89·8
4·9
547·8
136·8
23·5
40·1
456·9
27·3
6·9
13·9
104·9
H·l,
-~08-1- 2.02~
I
6·67
I
0·975
10.986
I
0·059
0·255
0·453
0·453
1·898
1·272
0·874
0·986
0·038
0·264
0·447
0·561
1·54
46·34
0·23
559
WHEAT GRAIN DEVELOPMENT
TABLE
Days I Grain
after
Part
Anthesis I
Total
Nitrogen
(I-'g)
Total
Protein
Nitrogen
(I-'g)
---1-- - - - - 1 - - - 36
I
W
R
E
G
-~----
42
W
0
R+E
G
Soluble
Protein
Nitrogen
(I-'g)
~---~
668·0
lii2·8
18·9
34·3
, 572·6
5·6
U·8
116'1
19·3
878·0
0
2 (Continued)
I
I
Chloro- Glutamyl
Acid
I TransPhosphyU
Transaminase
Content
ferase
phatase (I-'molesjhr)
(/lO.D.jhr) (/lO.D.jhr)
(I-'g)
4·52
_ _ _ 1_~2'_2 - - - - - - - - 1126·6
8159·9
167·1
15·7
786·0
158·2
4·0
140·0
23·1
---
----
2·09
1·042
1·121
69'&9
0·480
0·1512
0·032
0·018
0·237
0·294
0·516
0·074
3·33
66·56
0·636
1·2lO
69·98
0·291
0·320
0·025
0·155
0·9415
0'1l0
67·156
2·42
To illustrate the pattern of growth in the various tissues the total protein data
of the different parts were recalculated as percentages of the maximum values
attained by them, and are plotted in Figure 3. This diagram stresses the existence
of a series of growth cycles, each characteristic for a particular grain part. This was
first noticed by Nutman (1939) with respect to postfertilization phenomena in rye.
Indeed it seems that the concept can be extended even beyond maturation because,
during germination, the endosperm will also break down.
(c) Distribution of Enzyme Activities in Grain Parts
The enzymes assayed during a considerable period of the grain's development were: glutamyl transferase, acid phosphatase, and alanine-glutamic acid
transaminase.
All direct information obtained on these enzymes is again given in Table 2.
Absolute values on a grain basis are presented in a series of diagrams, paired with
diagrams illustrating distribution ratios (Figures 4, 5, and 7).
Specific activities, using the soluble protein data as a basis, are given in Table 3.
(i) Glutamyl Transferase.-This investigation constitutes the third harvest
series in which a peak of activity for the whole grain was found on the 12th day after
anthesis. As stated previously (Rijven and Banbury 1960), this peak is not an artefact
of extraction due to accumulation of inhibitors or of interfering enzymes. The peak
is explained by the finding that the enzyme is almost exclusively localized in the
future grain coat tissues, particularly in the outer pericarp. Although the activity
decreases after day 12, concomitantly with protein loss, this tissue remains the most
important contributor of glutamyl transferase activity (Fig. 4). As may be expected
the specific activity of this tissue is considerably higher than of any other. It is also
560
A. H. G. C. RIJVEN AND R. COHEN
TABLE
3
SPECIFIC ACTIVITIES OF ENZYMES IN PARTS OF DEVELOPING GRAIN OF WHEAT
Activities per mg soluble protein nitrogen: W = whole grain, 0 = outer pericarp, R = "rest of
coat", E = endosperm, G = germ
Days after
Anthesis
Grain Part
Glutamyl
Transferase
Acid Phosphatase
4
W
65·77
14·29
8
W
112·76
11·99
0
R
E
179·80
46·88
4·0
17·48
7 ·19
1·43
I
I
I
':rransaminase
--~~----
-------- --------
------
12
107·34
14·13
175·29
0
R
E
227·27
54·04
2·62
21·21
13·35
4·39
23·64
167·08
421· 50
W
44·77
13·41
524·54
0
R
E
180·0
49·25
1·07
32·25
17 ·71
5·43
61·67
219·40
822·70
W
31·41
12·04
502·31
0
R
E
136·02
59·09
1·30
33·73
17·68
5·82
27 ·12
89·89
634·34
9·81
406·33
W
I
---------
16
------20
----~~--
24
--~~----
W
17·06
0
R
E
G
139·29
59·04
0·66
-------
30
---------
----~----
-----~---
I
36·43
27 ·13
5·04
92·22
516·04
46·94
--------
W
13·87
9·30
0
R
E
G
126·67
70·91,
0·36
38·26
32 ·16
0' fi
-------- -------- ----
36
W
6·82
7·34
457·39
0
R
E
G
85·71
43·39
0·28
0·93
42·32
24·92
4·44
3·83
282·20
573·30
W
3·81
7·24
418·79
0
R+E
G
72·75
2·29
1·08
38·75
6·75
4·76
482·57
104·76
------------------42
561
WHEAT GRAIN DEVELOPMENT
interesting to observe that whilst total and soluble protein remain constant in the
outer pericarp between day 8 and 12, the activity of this enzyme increases there.
The relatively low activity of the endosperm fraction observed before (Rijven
and Banbury 1960) now appears to apply throughout development. The activity could
not be increased by dialysis.
(ii) Acid Phosphatase.-The distribution of this enzyme is much less specific,
and to some extent reflects the distribution of protein (Fig. 5). However, it may be
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DAYS AFTER ANTHESIS
Fig. 3.-Protein nitrogen content of grain parts. Data are expressed as per·
centage of maximum value ever attained by the part. The asterisk at day 0
marks protein content of the whole grain expressed as a percentage of the
maximum value attained by the outer pericarp. 0, outer pericarp; R, "rest
of coat"; E, endosperm; G, embryo (= germ).
noticed that the specific activity of the maternal tissues is significantly higher than
that of the endosperm. This may well be connected with the early breakdown of
these tissues. Another observation is that acid phosphatase activity remains high
in the outer pericarp for some time after day 12 whereas protein content and glutamyl
transferase activity fall off from then onwards. This is illustrated in Figure 6 by
expressing the activities as percentages of the maximum attained. It is also apparent
from this figure that throughout the period of physiological autolysis the relative
activity of acid phosphatase is higher than that of glutamyl transferase. The possible
significance of this pattern in relation to cellular enzyme localization will be considered
in Section IV.
562
A. H. G. C. RIJVEN AND R. COHEN
(iii) Transaminase.-Activity was found in all fractions, but the specific
activity was highest in the endosperm. This, together with the fact that much more
protein is accumulated there than anywhere else, makes the contribution of endosperm
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DAYS AFTER ANTHE51S
10
20
30
40
Fig. 4.-Distribution of glutamyl transferase activity in grain parts. (a) Additive plot in absolute
values. (b) Additive plot in ratios of the whole grain. 0, outer pericarp; R, "rest of coat"; E,
endosperm; G, embryo (= germ),
to total activity high throughout (Fig. 7). The activity in the outer pericarp was
low, in fact at the limit of detection possible by the methods employed. In the rest
of the grain coat the specific activity was definitely higher than in the outer pericarp.
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20
30'
40
10
DAYS AFTER ANTHESIS
20
30
40
Fig. G.-Distribution of acid phosphatase activIty in grain parts. (a) Additive plot in absolute
values. (b) Additive plot in ratios of the whole grain. 0, outer pericarp; R, "rest of coat";
E, endosperm; G, embryo (= germ).
The rise of activity in the rest of the grain coat on day 36 (Table 2) is probably due
to contamination by endosperm. The distribution of transaminase resembles that
of leucine-activating enzyme (Rijven and Banbury 1960).
563
WHEAT GRAIN DEVELOPMENT
(iv) Alcohol Dehydrogenase.-This enzyme was only assayed in extracts of
endosperms and of embryos of day 36. Endosperm showed higher activity on a grain
basis (~O.D.(hr = 21·84 for endosperm, 12 ·60 for embryo), but on a soluble protein
nitrogen basis the activity was higher in the embryo (~O.D .(hr = 652 . 8 per mg nitrogen
for embryo, 188·1 per mg nitrogen for endosperm).
IV.
DISCUSSION
Previous studies of changes in nitrogenous constituents in ripening seeds and
cereal grains have been reviewed by McKee (1958). In general it is recognized that
different proteins are synthesized at different stages and in particular that the reserve
proteins are synthesized during the later stages of development. In the present
100
PHOSPHATASE
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75
x
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GLUTAMYL
TRANSFERASE
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fO
20
DAYS AFTER ANTHESIS
30
40
Fig. 6.-Glutamyl transferase and acid phosphatase activity in outer
pericarp. Data expressed as percentage of maximum value attained therein.
investigation histological aspects of grain development have been taken into account
and it is now established that the above feature, concerning the development of the
whole grain, also holds for an aspect of protein synthesis in an important grain
part, the endosperm. It can be readily established from Table 2 that up to day 16
soluble protein nitrogen contributes about 40% of the total protein nitrogen content
of the endosperm; thereafter this percentage decreases until finally it is less than
20 % of the total.
A peculiar fact revealed in these studies is the distribution pattern of glutamyl
transferase. Comment has been made already on this fact in connection with glutamine
synthesis in the developing grain and the possible metabolic role of the coat tissues
(Rijven and Banbury 1960).
It may be emphasized now that the localization of this enzyme is not unique
for wheat and that an essentially similar distribution has been found in barley and
in a very different plant species, viz. pea (Rijven 1961b). In the developing pea seed
the specific activity of the embryo is many times lower than that of the seed coat.
In attempting to explain this phenomenon, it is considered of importance· to
stress that in both cases the development of the maternal tissue parts precedes that
564
A. H. G. C. RIJVEN AND R. COHEN
of the enclosed endosperm or embryo, and that the latter receive all their substrates
from the maternal tissues. Further, attention must be drawn to observations which
show that glutamyl transferase synthesis is repressed by glutamine. This has already
been shown for HeLa cells by De Mars (1958). Using excised germinating wheat embryos
it has now been established (Rijven 1961a) that exogenous glutamic acid stimulates
synthesis of the enzyme to the same degree as protein synthesis. Glutamine, however,
although stimulating protein synthesis, does not give a glutamyl transferase level
beyond that of a control-no nitrogen-treatment, when it is ltPplied in the range
of 10-50 mM concentration. Addition of mixtures of glutamine and glutamic acid
results in intermediate enzyme levels.
1·00
BO
(a)
-;;
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bI 40
III
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i
Z
I-
20
o
o
10
20
30
10
40
AFTER ANTHESIS
20
30
40
DAY~
Fig. 7.-Distribution of alanine-glutamic acid transaminase in grain parts. (a) Additive plot in
absolute values. (b) Additive plot in ratios of the whole grain. 0, outer pericarp; R, "rest of
coat"; E, endosperm; G, embryo (= germ).
These facts lead to the speculation that, in the grain or seed, a high glutamyl
transferase level may become established in the earlier developing maternal parts
because, in the incoming stream of nutrients, the amount of glutamic acid exceeds
that of glutamine. Thus a "barrier" of glutamyl transferase may be established,
preventing the passage of glutamic acid into the interior in amounts more than a
fraction of the glutamine present, as during the course of its passage, glutamic acid
would be converted into the amide by the enzyme. Hence in the interior, in endosperm or embryo, repression of the synthesis of the enzyme would follow. This
hypothesis is not inconsistent with the observation that, upon germination the level
of glutamyl transferase activity rises rapidly in both endosperm and embryo (Rijven
1961a). For in germinating barley, and presumably also in wheat, glutamic acid
outweighs glutamine (labile amide) in the "soluble nitrogen" pool (Folkes and
Yemm 1958). However, free glutamic acid is also present in the isolated developing
endosperm at a concentration of at least 2·5 mM (Rijveil, unpublished data). It
would seem that to maintain the present explanation,compartmentalization of
amino acid pools (Steward, Bidwell, and Yemm 1958) would be necessary.
WHEAT GRAIN DEVELOPMENT
565
Another observation of interest is that in the outer pericarp, acid phosphatase
activity remains high after day 12, whilst glutamyl transferase activity and total
protein content fall off (Fig. 6). Strikingly similar patterns of enzyme ontogeny
have been observed during the autolysis of animal tissues, and their features have
been embodied within the lysosome concept (de Duve 1958). There exist subcellular
particles, lysosomes, containing a group of hydrolytic enzymes (amongst them acid
phosphatase) which can break down cell constituents of major importance. However,
the authors were unable to find evidence for the existence of such particles in young
developing grains using osmotic shock or "Triton X 100 " treatment of extracts. An
alternative solution to the localization and role of these enzymes is indicated by the
finding of Lamport and Northcote (1960) that acid phosphatase activity of plant
tissue culture material was largely bound up with a cell wall preparation suggesting
it to be a surface enzyme.
With respect to the data on transaminase and alcohol dehydrogenase little
comment seems necessary, but it may be pointed out that they provide evidence
for biochemical differentiation; for anyone of the parts studied it is now possible
to mention an enzyme showing highest specific activity there. This does not apply
strictly to the rest of the coat but its unique high chlorophyll content implies a whole
complement of enzymes.
V.
ACKNOWLEDGMENTS
The authors are indebted to Drs. R. F. Williams and D. Spencer of this
Division for discussion and advice. Messrs G. A. McIntyre and G. N. Wilkinson,
Division of Mathematical Statistics, C.S.I.R.O., helped devise the sampling technique.
VI.
REFERENCES
ARNON, D. 1. (1949).-Plant Physiol. 24: 1.
AXELROD, B. (1947).-J. Biol. Chem. 167: 57.
COHEN, P. P. (l955).-Estimation of animal transaminases. In "Methods in Enzymology".
Vol. 2. p.178. (Eds. S. P. Colowick and N. O. Kaplan.) (Academic Press Inc.: New York.)
COWGILL, B. W., and PARDEE, A. B. (1957).-"Experiments in Biochemical Research Techniques."
(John Wiley and Sons Inc.: New York.)
CRUICKSHANK, D. H., and ISHERWOOD, F. A. (1958).-Biochem. J. 69: 189.
DE MARS, R. (1958).-Biochim. Biophys. Acta 27: 435.
DUVE, C. DE (1958).-Lysosomes, a new group of cytoplasmic particles. In "Subcellular Particles".
p. 128. (Ed. T. Hayashi.) (Ronald Press Co.: New York.)
FOLKES, B. F., and YEMM, E. W. (1958).-New Phytol. 57: 106.
KRAUSS, L. (1933).-Jb. wiss. Bot. 77: 733.
KUDELKA, E. (1875).-Landw. Jb. 4: 241.
LAMPORT, D. T. A., and NORTHCOTE, D. H. (1960).-Biochem. J. 76: 52P.
McKEE, H. S. (1958).-Protein metabolism in ripening and dormant seeds and fruits. In
"Encyclopedia of Plant Physiology". Vol. 8. p. 581. (Ed. W. Ruhland.) (Springer
Verlag: Berlin.)
NUTMAN, P. S. (1939).-Ann. Bot., Lond. (N.S.) 3: 731.
PERCIVAL, T. (1921).-"The Wheat Plant." (Duckworth & Co.: London.)
566
A. H. G. O. RIJVEN AND R. OOHEN
RACKER, E. (1955).-Alcohol dehydrogenase from baker's yeast. In "Methods in Enzymology".
Vol. 1. p. 500. (Eds. S. P. Colowickand N. O. Kaplan.) (Academic Press Inc.: New York.)
RIJVEN, A. H. G. C. (1961a).-Biochim. Biophys. Acta (in press).
RIJVEN, A. H. G. C. (1961b).-Aust. J. Biol. Sci. 14: 700.
RIJVEN,A. H. G. C., and BANBURY, C. A. (1960).-Nature 188: 546.
STEWARD, F. G., BIDWELL,R. G. S., and YEMM, E. W. (1958).-J.
Exp. Bot. 9: 11.
STUMPF, P. K., LOOMIS, W. D., and MICHELSON, C. (1951).-Arch. Biochem. Biophys, 30: 126.
EXPLANATION OF PLATE
1
Fig. I.-Transverse section at low magnification through a grain of wheat harvested 12 days
after anthesis. 0, outer pericarp: ep., epidermis on the outside; par., parenchyma with
starch granules; S, schizogenous gap separating outer pericarp from inner pericarp;
R, rest of coat: v, vascular bundle. E, endosperm with large cells showing mitotic
activity, but with no starch present as yet.
Fig. 2.-Details at high magnification of a part similar to, but not identical with, the rectangle
shown in Figure 1. S, R, and E as.for Figure 1; chl., chlorophylliayer-in vivo the cells
of this layer -are packed full with distinct chloroplasts at this stage; t, inner epidermis
of pericarp-components are torn asunder, particularly on the lateral sides, and are
known as "tubule cells"; i.i., inner integument of two cell layers showing mitotic
activity; n, epidermis of nucellus.
RIJVEN AND COHEN
PLATE
WHEAT GRAIN DEVELOPMENT
Aust. J. Biol. Sci., Vol. 14, No.4
1