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Wheat Information
Service
Number 76: 73-76 (1993)
Comparative
phylogenetic study in amylase isozymes in Gramineae: Why are these
enzymes nice marker for phyIogenetical approach in common wheat?
H.
Ishihara1, Y. Furuta1, K. Nishikawa2
and N. Watanabe1
1Gifu University, Yanagido1-1, Gifu 501-11, Japan
2Aichi Sangyo University, Okamachi12-5, Okazaki 444,
Japan
Amylase plays a key role in metabolism of the plants, especially
during germination of starchy seeds. The molecular variants of this
enzyme have brought about the nice markers for phylogenetical
approaches in wheat, because they are genetically polymorphic and can
be detected easily by polyacrylamide isoelectrofocusing (Nishikawa
and Nobuhara 1971, Nishikawa 1983, Nishikawa et al 1992). On the
other hand, rice seed at germinating stage had only few isozyme bands
or less variable in comparison with wheat seed. While corn is
intermediate in the number of isozyme bands and variability. That is,
there are apparent interspecific: differences in some traits of
amylase isozymes in family Gramineae, which is dynamically
differentiates (Takeoka 1959) and contains several important cereals.
This brought us the vivid interests in differentiation of amylase
isozyme genetic system in the course of evolution of this large
family.
Materials and methods
Seeds of common wheat, oat, finger millet, corn and rice in three
conditions, dry, germinating and developing stages were used. The
following treatments were applied to the crude extracts of amylase
from respective seeds; (1) incubation in 70oC hot water
for inactivating beta-amylase, (2) dialyze in acetate buffers (pH
5.0-3.0) for investigating the critical pH of alpha-amylase tolerance
to acidity, (3) pappain or 2-mercaptoethanol treatment for activating
the latent beta-amylase. The isozymes were separated by the thin
layer polyacrylamide isoelectrofocusing.
Results and discussion
Table1
shows the characteristics of both aipha- and beta- amylase isozymes
in common wheat. It is apparent fromTable1
that there are genetical, physiological and biochemical
differentiation of these isozymes in wheat. Such differentiation of
the isozymes would he expected in the other cereals. So, five crops
were compared with one other in regard to these characteristics. The
results obtained are shown in Table
2. Hexaploid
wheat had 15 bands of beta-amylase in dry seed, oat and corn having a
single band of beta-amylase. Whereas millet and rice had none of
beta-amylase band in dry seed. BY the treatment of pappain, 14 bands
of beta-amylase isozymes were induced in wheat, and eight bands in
corn. But none was induced in oat and rice. Unfortunately, millet was
out of analysis. Three to 22 bands of beta-amylase isozyme were found
in germinating seed of these five cereal plants. In wheat, 12 bands
of out of these found during germination also occurred in developing
seed, five bands in oat and two bands in corn, respectively.
Beta-amylase isozyme bands in wheat and oat were tolerant to the
stronger acidity (pH 3.0), whereas the critical pH for
those in corn and rice was 3.6.
As previously reported, 15 malt type bands and nine green type bands
of alpha-amylase isozyme were detected in germinating seed of common
wheat. Ten, six, twelve and four alpha-amylase isozyme bands were
detected in germinating seed of oat, millet, corn and rice,
respectively. Three very faint bands occurred in the early stage of
developing seed in wheat. Oat, corn and rice had five, two and three
faint bands in the young developing seed, respectively. Malt type and
green type of alpha-amylase isozymes in wheat are different in
several characteristics as shown in Table1
. The critical pH of acid tolerance was 3.6 for malt type isozymes
and 3.0 for those of green type in wheat, respectively. Alpha-amylase
isozymes in oat, millet, corn and rice were inactivated at pH 4.0,
5.0, 3.6 and 4.0, respectively. These pH data were not sensitive
enough for clear discrimination into two types of isozymes. However,
Gonokami et al (1992) reported clear discrimination of alpha-amylase
isozymes of wheat into malt and green types by use of differential
adsorption ability of the intact raw starch granules. They also could
detect four of eleven bands of corn and two of three bands in rice as
malt type. The result suggests that malt type isozymes tend to
distribute in the higher pH zone in these materials as in the case of
wheat.
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