Plant Physiol. (1991) 96, 84-90
0032-0889/91/96/0084/07/$01 .00/0
Received for publication October 9, 1990
Accepted December 17, 1990
Independent Regulatory Aspects and Posttranslational
Modifications of Two ,-Amylases of Ryel
Use of a Mutant Inbred Line
Jean Daussant*, Jan Sadowski, Tadeusz Rorat, Christiane Mayer, and Christiane Lauriere
Laboratoire de Physiologie des Organes VWg6taux, CNRS, 4 ter route des Gardes, 92190 Meudon, France
(J.D., C.M., C.L.); and Institute of Plant Genetics, Polish Academy of Sciences, 30/36 ul. Strzeszynska,
60-479 Poznan, Poland (J.S., T.R.)
that cereals contain different types of f3-amylase which may
have different physiological functions.
We aimed at further investigating the differential expression
of f3-amylase in cereals with regard to regulatory aspects such
as relative abundance, tissue location, and time sequence of
accumulation/disappearance. To do this we analyzed p3-amylase expression in kernel and vegetative tissues of a normal
inbred line of rye in comparison with a mutant line having
been previously shown to be deficient in the enzyme (5). Such
a comparison should enable the detection of functionally
important features of differential expression of f3-amylase
enzymes in cereals, especially with regard to the role of
posttranslational modification.
ABSTRACT
We have examined the occurrence/disappearance, tissue location, and posttranslatlonal modification of j-amylase proteins
in rye (Secale cere L.) kernels at three physiological stages
(development, maturity, germination) with a normal inbred line
and a mutant line exhibiting a high but incomplete fl-amylase
deficiency. This deficiency corresponds to a lack of accumulation
of fl-amylase activity in the endosperm and does not affect the
level of activity in the outer pericarp and green tissues as compared to the normal line. Two antigenically related but distinct ,amylases (I and 11) were detected in the normal line (II being the
major constituent) and only one (I) in the mutant line. I and 11
display very similar electrophoretic polymorphism. In both lines,
I appears to be ubiquitous, although it disappears from the outer
pericarp during ripening. Antigen II was present only in the normal
line and appears to be specific for the endosperm and perhaps
for the maternal green tissues of the seed. Posttranslational
modifications occurring during germination, which are mimicked
by the action of papain, affect 11 but not 1. The two groups of 0amylases are discussed in relation to recent reports indicating
the presence of two types of j0-amylase with different functions
and gene loci in barley and wheat.
MATERIAL AND METHODS
Plant Material, Dissection, and Extraction
Two lines of rye (Secale cereal L.) obtained from the
laboratory of rye genetics, Academy of Agriculture, Cracow,
were used in this study: One "normal" line (Kazimierskie B4)
and one mutant line exhibiting a high deficiency in fl-amylase
activity (Wegierskiel 1). Plants were grown in greenhouses in
Poznan in 1989. Seeds were collected at 10 and 24 d after
anthesis and at maturity (between 50 and 60 d after anthesis).
The samples were immediately frozen in liquid nitrogen and
kept at -80'C, except for the seeds taken at maturity which
were stored at 20C. To obtain germinated seeds and leaves,
seeds were surface-sterilized with 1% NaOCl and sown on wet
vermiculite. Germination and growth were carried out at 230C
under 12 h light (12 W. m-2) per day. Leaves were collected
7 d after sowing and kept frozen.
The colorless outer pericarp derived from the ovary wall
(14) was easily removed from developing and presoaked mature seeds (3). After removal of the outer pericarp, the green
tissues (testa and the nucellar tissues [14]) were separated
from the developing seeds or scratched out from the mature
seeds. The green tissues prepared from mature seeds were
highly contaminated with pieces of the endosperm.
Enzymes were extracted from whole organs and from dissected tissues in a mortar. Grinding was intermittent for 30
min at room temperature. The proportion of tissue to extraction medium (0.1 M NaCl) was 100 ,ug-mL-' for the seed
fl-Amylases in barley, wheat, and rye kernels are known to
consist of several distinct constituents, which nevertheless are
antigenically identical and which exhibit a common developmental phenomenology. The enzyme is synthesized in the
endosperm of developing seeds and is stored partly in a bound
form which is then posttranslationally modified during germination. The modifications involve proteolytic cleavage
(1, 2, 8, 12, 15, 19).
Recent reports indicate, however, the presence of fl-amylase
activity not only during the early phases of kernel development but also in other organs of barley (10) and wheat (3)
seedlings. In addition, two loci encoding for fl-amylase have
been identified (10) and two related but distinct fl-amylase
antigens have been characterized (3). These findings suggest
'The study was carried out in the framework of the exchange
program between the Centre National de la Recherche Scientifique,
France, and the Polish Academy of sciences.
84
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REGULATORY ASPECTS AND PROCESSING OF RYE f-AMYLASES
tissues and 1 g mL-' for the leaves. The homogenates were
centrifuged at 13,000 g for 10 min. The extracts were analyzed
either immediately after preparation or after being kept overnight at 4C. It was determined in preliminary assays that
extraction at room temperature or overnight storage of the
extracts at 40C resulted in activity levels and IEF2 patterns
identical to those obtained with fresh extracts prepared at 40C.
85
in the mutant line than in the normal line 24 d after anthesis
(Fig. 1). In both lines, the activity almost vanished at maturity.
In the endosperm and the green tissues of the normal line,
the f3-amylase activity increased during ripening, whereas, in
the mutant line, endosperm and green tissues were almost
devoid of activity at maturity. In the leaves of 7-d-old seedlings
(normal and mutant), the activity levels found were very low
and similar to that found in mature mutant kernels.
Immune Serums
Two anti-barley a-amylase immune serums were used for
removing the high and low pI rye a-amylases from the extracts
during IEF (1, 3). An anti-barley fl-amylase immune serum
was used for characterizing the rye f3-amylase. As previously
reported with respect to the characterization of another immune serum (1), this immune serum was shown to react with
all forms of f3-amylase found in rye seeds and leaves. The
immune serum was further tested for its monospecificity by
means of immunoelectrophoretic analysis (7): various combinations of extract and serum concentrations yielded always
only one immunoprecipitin band upon protein staining. This
precipitin band had j3-amylase activity.
Analytical Techniques
a- and fl-amylase activities were assayed using synthetic
substrates as previously described ( 18).
Rocket-line immunoelectrophoresis ( 11) was carried out in
1.2% agarose gel as previously described (3) under conditions
specified in the legend of Figure 3. Wheat pericarp and
endosperm extracts from seeds at 24 d postanthesis were used
for constructing the immunoprecipitin lines corresponding to
f3-amylases I and II which build up during electrophoresis.
The pericarp and the endosperm extracts were diluted 4 and
24 times, respectively. Electrophoresis was carried out at
40C and 5 V cm-' for 15 h. After electrophoresis the gels
were stained for amylase activity using soluble starch as
substrate (5).
Isoelectric focusing was carried out in 2 mm thick 6.5%
polyacrylamide gel containing 5% Ampholines, pH range 4
to 8 (LKB Broma, Sweden). Extracts were applied to the
cathodic side of the gel and electrophoresis was started without
preforming the gradient. IEF was combined with immunoabsorption (4) to specifically remove a-amylases which interfere
with f3-amylase staining. Staining for a- and for both a- and
,B-amylases was carried out with 3-limit dextrins and soluble
starch, respectively, as substrates (4).
In the papain treatment, 2.5 mg papain (Sigma) were added
to 0.5 mL of extract. Extracts were stirred gently and left at
20°C overnight. Blanks were obtained by incubating untreated
extracts under the same conditions.
Electrophoretic Polymorphism
Amounts of extracts possessing similar activities were applied to the gels (Fig. 2). In these experiments, a-amylase
present in the extracts of developing kernels and leaves was
removed by electroimmunoabsorption (1). The effectiveness
of this immunoelimination is shown in Figure 2A. The same
IEF diagrams were obtained with fresh and stored extracts
indicating that none of the heterogeneity observed was due to
storage artifacts as was the case for the unstable debranching
enzyme extracted from spinach leaves (13).
The zymograms of the extracts of the three investigated
tissues of the mutant kernels sampled at 24 d were qualitatively identical (right part of Fig. 2, B 1). In kernels of the
normal line sampled at the same stage, some qualitative and
quantitative differences were evident between the zymograms
of the three tissues (left part of Fig. 2, B 1). All the fl-amylases
present in the mutant line were found in the normal line,
whereas additional fl-amylases were detected in the normal
line. The zymograms of the tissue extracts of the mutant
kernels sampled at maturity were all similar (right side of Fig.
2, B2), and were also similar to zymograms of extracts of
124 dm
10
8
F24
We~
Activity
In the outer pericarp, the ,B-amylase activity was highest 10
d after anthesis in the normal line and was significantly higher
2 Abbreviations: IEF, isoelectric focusing; pI, isoelectric point; IgG,
immunoglobulin G; IS, immune serum.
~
~
~
4d
I
0
"I3
08
3
01
01
l!1 S!1.0 dell~ ,.11
1l
III II
00
P G E
RESULTS
IMatur.
P
G
E
P G E
|INORMAL
L
P
G
E
P G E
L
[MUTANT|
Figure 1. fl-Amylase activity in tissue extracts of developing seeds,
mature seeds, and 7-d-old leaves of a normal and a mutant rye line.
The activities quoted represent those present in the tissues of a
single whole organ (seed or leaf). P, outer pericarp; G, green tissues;
E, endosperm; L, leaves. Two to three series of experiments were
carried out. An arrowhead indicates the sample series used for the
experiments reported in Figure 3.
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86
DAUSSANT ET AL.
Plant Physiol. Vol. 96, 1991
24 days after anthesis
A
NL
1
MP
1.6
BI
ML
1.3
NP NG
1
1
NE
1.6
C
MP
1.6
MG
1.6
ME
0.9
NL
1.4
MP
1.6
ML
1.3
PH8
pH4
Mature
B2
NP
2
NG
2
NE
1.8
MP
2
MG
2
ME
1.9
+
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REGULATORY ASPECTS AND PROCESSING OF RYE fl-AMYLASES
mutant kernels sampled at 24 d (right side of Fig. 2, B 1). The
IEF patterns of the tissue extracts of the normal kernels
sampled at maturity (left side of Fig. 2, B2), differ from those
of the 24 d kernels (left side of Fig. 2, B1) by the increasing
number and relative intensity of constituents having high pI
values. The zymogram of the outer pericarp extract from the
mutant line and those of the leaf extracts from the two lines
were qualitatively identical with the exception of one low pI
leaf constituent (Fig. 2C).
Antigenical Identification
Preliminary experiments with the anti-barley fl-amylase
immune serum revealed two antigenically related but distinct
fl-amylases in the rye kernels. One antigen (I) reacts with only
some of the antibodies contained in the immune serum,
whereas the other antigen (II) reacts with a larger set of
antibodies of the same immune serum. Line immunoelectrodiffusion was used to identify each antigen in the tissues (Fig.
3). Two precipitin lines corresponding, respectively, to antigen
I (upper line) and antigen II (lower line) were observed. The
presence of a peak above one line indicates the presence of
the antigen in the extracts deposited in the wells. The sizes of
the peaks are positively related to the amount of deposited
antigen.
Antigen I was detected in all tissues of the normal 24 d
kernels (Fig. 3, upper part). It appeared in similar concentration in the pericarp and in the green tissues and in about 10fold higher concentration in the endosperm extract. Concentration of antigen II is the highest in the endosperm extract,
10 times smaller in the green tissue extract, and practically
absent in the extract of the outer pericarp. Between 24 d
development stage and maturity, antigen I vanished from the
outer pericarp and antigens I and II slightly decreased in the
endosperm. Taking into account the respective weights of the
tissues at the mature stage (endosperm: 25.7 mg; green tissues,
highly contaminated with the endosperm: 4.7 mg; outer pericarp: 1 mg) it can be concluded that at least 80% of each
antigen is located in the endosperm. Eighty to 90% of
the activity was also found to originate from the endosperm
(Fig. 1).
In the mutant line no antigen II was found in any tissues
from the developing or mature kernels. In the developing
kernels antigen I was found in the pericarp and in the green
tissues but not in the endosperm. This is probably due to the
very low activity found in the extract of the endosperm (0.1
units/mL) compared to that found in the outer pericarp and
in the green tissue extracts (1 1.2 and 17.9 units/mL, respectively). The significant total activity found in the endosperm
(Fig. 1) is due to the larger amount of this tissue (27 mg)
compared with those of the green tissues (3 mg) and of the
87
outer pericarp (4 mg). The changes respective of antigen upon
maturation reflect the changes in activity (Fig. 1), and are
similar to those observed with the normal line. The results
indicate that the mutation concerns solely antigen II.
fl-Amylase in the leaf extracts was identified as antigen I in
both the normal line (Fig. 3) and in the mutant line (not
shown).
Posttranslational Modifications of 18-Amylase during
Germination of the Normal and Mutant Kernels
Mutant and normal rye line kernels were grown under the
conditions. After 5 d, seedlings with about 3 cm long
shoots (from both the normal and mutant lines) were sampled.
The kernels were separated from the shoots and roots and
extracted. The amylase activities (a and (3) of the extracts were
measured (Table I). For both lines the a-amylase activity was
considerably higher in the germinated than in the ungerminated kernels. The level of fl-amylase activity in the kernels
of the normal line increased slightly upon germination: this
corresponds to the conversion of the bound form of the
enzyme into the free form (1). However, the fl-amylase activity
remained at a very low level and even decreased in the mutant
line kernels.
During germination, rye fl-amylase of the normal line kernels is subjected to modifications, resulting in the disappearance of low pI constituents and in the appearance or increase
in the amount of other constituents with higher p1 values (left
part of Fig. 4). No such modifications were observed for the
kernels of the mutant line. The low pI constituents of mutant
kernels showed no changes in pI values during the course of
same
germination.
Because such posttranslational modifications (1) are known
19), we examined
the effect of papain action on the ,B-amylases extracted from
the ungerminated kernels of both lines (right part of Fig. 4).
With respect to the normal line, new constituents with high
pI values appeared or increased in intensity, whereas the
constituents with low pI values vanished. In contrast, for the
mutant line, if low pI constituents faintly appear, the bulk of
the low pI constituents nevertheless remained unmodified.
These results indicate that antigen I, the only antigen to be
detected in the mutant line, is much more resistant to the
action of papain than antigen II, which is the main constituent
in the normal line.
to be mimicked by the action of papain (1,
DISCUSSION
As is the case for wheat kernels (3), two fl-amylase groups
designated I and II could be distinguished in rye kernels
according to differences in antigenicity. Several constituents
Figure 2. IEF of organ extracts. a-Amylase was specifically removed by immunoelimination. Papers soaked in the IgG fraction of the anti-aamylase immune serum were first layered onto the polyacrylamide gel, and papers loaded with the extracts to be investigated were placed
directly upon them. Different amounts of the extracts or of their dilutions (10-60 uL) were applied. The numbers indicate the enzyme activity
deposited (units = number x 1 0-). N, normal line; M, mutant line; L, leaf; P, outer pericarp; E, endosperm; G, green tissues. Enzymatic activity
present after completion of IEF was characterized with ,j-limit dextrins (A) and with starch (B and C) as substrates. The arrow in A indicates the
presence of a blue band on the red background which corresponds to the activity of the debranching enzyme. Note that no a-amylase activity
was apparent in the gel after immunoelimination (A).
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88
DAUSSANT ET AL.
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Plant Physiol. Vol. 96, 1991
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89
REGULATORY ASPECTS AND PROCESSING OF RYE g-AMYLASES
Table I. Characteristics of Ungerminated and Germinated Kernels of the Normal and Mutant Lines
Kernels were sampled after 5 d of germination and growth. The values reported are the mean values
obtained with samples of eight kernels. Standard deviation is indicated between brackets.
Mutant Line
Normal Line
Ungerminated
Germinated
Ungerminated
32.9 (5.1)
36.4 (4.1)
3 (0.9)
5.6
(1.9)
8.9
(3.5)
18.4 (3.3)
Kernel weight, ga
Shoot length, cm
a-Amylase activity, units
0.006
(0.0034)
6.2
,3-Amylase activity, units
(2.6)
a
The kernels were weighed before soaking.
0.01
(0.007)
0.056
(0.03)
Germination
N
U
Papain
M
5
U
Germinated
19.4 (2.9)
3.1 (0.7)
5.8
(1.7)
0.04
(0.009)
N
5
U
M
U+P
U
U+P
pH8
+
pH4
Figure 4. Modification of fl-amylases occurring naturally in kernels during germination or induced by treatment of extracts of ungerminated
kernels with papain. The extracts were analyzed by IEF. N, normal line; M, mutant line; U, extract of ungerminated kernels; 5, extracts of 5-d
germinated kernels; U + P, extracts of ungerminated kernels after incubation with 0.5% papain. The amounts of the extracts of their dilutions
applied (10-60 uL) were adjusted so that the activity applied in each case was between 1 and 2 x 1 -2 units.
or
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90
DAUSSANT ET AL.
with differing pI values compose each of I and II. However,
the constituents of antigen I have pI values very close to those
of group II constituents.
The mutant line is highly deficient in f3-amylase having less
than 1% of the activity present in the normal line kernels.
This deficiency is due to a lack of enzymatic protein synthesis
(5). In a study on barley mutants exhibiting either a large
excess or deficiency in f3-amylase activity, it was shown that
two types of mRNA were involved in the synthesis of the
endosperm fl-amylase and that the excess or the deficiency
was due to a modulation in the endosperm mRNA synthesis
(9). Moreover, the mutation did not affect the expression of
f3-amylase mRNA in other organs, i.e. leaves and shoots. In
the present study, the deficiency was found to be due to the
lack of antigen II in the developing and mature kernels,
whereas antigen I was not significantly affected. This indicates
that the bulk of the activity, in the normal line, is due to
antigen II. Moreover, the mutation did not affect the level of
the f3-amylase activity in either the outer pericarp or the green
tissues or the leaves. Only antigen I was identified in the leaves
of both the normal and the mutant lines. These results indicate
that f3-amylases I and II correspond to different isozymic
groups which are subject to different regulatory mechanisms.
The independence of the regulatory features of f3-amylases
I and II suggests that the two groups have different functions.
fl-Amylase I appears to be ubiquitous since it was found in
all investigated tissues, including those exhibiting catabolic
(pericarp) and assimilatory (leaves) metabolisms. The endosperm f3-amylase (II) is supposed to be functionally important
during germination, where it could serve either as an enzyme
and/or as a storage protein (5, 6, 17). The inferred function
as a storage protein may be specific for this enzyme. Indeed,
during germination, a proteolytic enzyme cleaves a peptide
of about 5 kD from the f3-amylase in barley (19), wheat and
rye (1). As this splitting did not appear to have any significant
consequence for the specific activity of the enzyme (19), the
physiologically important result of the splitting may be the
liberation of the 5 kD peptide. The lack of posttranslational
modification of the fl-amylase of the mutant line can be
ascribed either to a lack of proteases affecting the modification
and/or to the insensitivity of antigen I to such proteases. The
second possibility was verified by the experiments with papain. This enzyme has been shown to mimick the posttranslational modifications undergone by ,B-amylase during the
germination of barley, wheat, and rye (1, 19) when added to
extracts of ungerminated kernels. The experiment carried out
with the mutant line, which contains antigen I only, showed
that the antigen resisted the papain action. This emphasizes
the structural difference between antigens I and II. It shows
furthermore that antigen I cannot fulfill the particular storage
protein function suggested for antigen II. In any event, ,3amylase II does not appear to be essential for the processes
involved during germination and early growth, since the
seedlings arising from mutant and normal line kernels appear
Plant Physiol. Vol. 96, 1991
to be very similar 5 d after the initiation of germination (Table
I and ref. 19).
ACKNOWLEDGMENT
The authors thank Dr. P. Ziegler (Universitiat Bayreuth, FRG) for
useful suggestions during the writing of the text.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
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