jag.50.225

225
J. Appl. Glycosci., 50, 225-230 (2003)
© 2003 The Japanese Society of Applied Glycoscience
Proceedings of the International Symposium: New Approaches in Starch Science and Carbohydrate-Active Enzymes
Session I: Structure of Starch and Its Biosynthesis
Isolation
and
Characterization
of Starch
(Received September
Mutants
in Rice
15, 2002)
Hikaru Satoh,1,*Aiko Nishi,1 Naoko Fujita,2,3Akiko Kubo,2,3Yasunori Nakamura,2,3
Tsutomu Kawasaki4 and Thomas W. Okita5
1 Faculty of Agriculture, Kyushu University (6-10-1, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
)2Faculty of Bioresource Science
, Akita Prefectural University (241-7, Shimoshinjo-Nakano, Akita 010-0195, Japan)
3CREST, JST, Japan
4Graduate School of Biological Science
, Nara Institute of Science and Technology (8916-5, Takayama, Ikoma 630-0101, Japan)
5nstitute of Biological Chemistry, Washington State University (Pullman, Washington 99164-6340, USA)
Abstract: We isolated various kinds of mutants participating in the biosynthesis of endosperm starch by the
MNU treatment of fertilized egg cells in japonica rice cultivars. There were at least 6 mutant loci lowering
amylose content including the wx locus. These low amylose mutations altered the amylose content without af
fecting the amylopectin structure. The amylose content did not affect the initiation of gelatinization, but influ
enced greatly the swelling of gel, indicating that amylose content affects the swelling power of gel and the termination of gelatinization. BEIIb mutation decreased specifically the short chains with DP less than 17 in
amylopectin whereas BEI mutation was characterized by a significant decrease in long chains with DP longer
than 38 and short chains with DP 12 to 23, suggesting that BEIIb and BEI contributes to the synthesis of A
chains, and B, and B3 chains of amylopectin, respectively. Notable alteration on the chain length profile was
not observed in BEIIa mutation. Each BE mutation showed distinct effect on the thermal properties of en
dosperm starch. The sug2 mutation lowering the expression level of PUL increased shorter chains of amy
lopectin and accumulated a significant amount of the water-soluble polysaccharides as well as ISA (sugl ) mu
tation. The fl o2 mutation decreased simultaneously the expression levels of some of the enzymes including
BE!, PUL and GBSS, suggesting that the wild type Flo2 gene probably encodes a regulatory protein that
modulates the expression of the genes involved in starch biosynthesis. Some mutants involved in the biosyn
thesis of ADP-glucose were also isolated.
Key words: starch mutant, low amylose, starch branching enzyme, starch debranching enzyme, ADPGPPase
Improvement of starch properties is one of the most important goals in
greatly influence
in addition to its
the purpose, the
rice breeding because starch properties
the eating and cooking qualities of rice
processing method for industrial use. For
genetic resources for improving starch
properties must be generated, collected, characterized and
evaluated. In maize, various kinds of mutants for polysaccharides reserves in the grain such as waxy, sugary,
shrunken and amylose-extender
have been identified.
These mutants have greatly contributed to the improvement of starch quality of maize, and thus expanded the
use of maize not only as food but also for important materials for food industry. In addition to the importance of
improving grain quality, maize mutants have provided
valuable information on gene action in metabolic regulation of the biological processes in higher plants. The
proper understanding of the processes will accelerate the
development of novel genetic resources for improving
starch properties.
We treated the fertilized egg cells of rice with Nmethyl-N nitorosourea
(MNU)1) and obtained several
thousands mutations for embryo and endosperm properties2'3' as numerous as those in maize. Using these NMUinduced mutants, the genetics and the effects of specific
*Corresponding author (Tel ./Fax. + 81-92--642-3056, E-mail: hsatoh
@agr.kyushu-u.ac.jp).
genes on qualitative and quantitative
changes in carbohydrates in rice endosperm are now being investigated.
Induction of mutations for endosperm properties of
rice by MNU treatment.
When the fertilized egg cells were treated with 0.75
mM MNU for an hour at different stages after flowering,
many mutants for embryo or endosperm properties were
induced as shown in Table 1. The mutation frequency
considerably differed among mutant types, the highest being white core mutant. This might be due to the difference in number of genes for each character, rather than
the difference in mutation frequency per locus for each
character. MNU mutagenesis being coupled with the fertilized egg cell treatment is characterized by high mutation frequency and a large size of mutated sector, and
thus many mutations were easily detectable. In addition,
due to the specificity of mutagenesis of MNU, most of
the mutants induced by MNU treatment were pointmutations, not deletion type as found by gamma irradiation or EMS treatment.4)So far more than two thousands
mutant strains have been screened and stocked in our
laboratory.
Mutants modifying amylose content of endosperm
starch.
Endosperm starch of japonica rice consists of about
J. Appl. Glycosci., Vol. 50, No. 2 (2003)
226
20%
to
amylose
and
amylopectin
properties
After
of cooked
treatment
of
mutants
with
fine
classified
amount
of
In
amylose
in
type
was
white.
was
mutants
tween
starches
stained
light
had
hazy
white
waxy
rice
which
Amylose
lines
were
ranging
dull
the
non-waxy
between
the
wx
ing
of
in
from
mutants
phenotype
locus,
amylose
3.2%
were
and
dull
waxy
and
five
other
contents
Table
amylose
non-waxy
solution,
The
there
content.
loci
endosperm
1.
Spectra
bemutant
iodine
or
solu-
waxy
among
rice.
mutant
phenotypes
of
between
of
wx
(dull)
dull
the
varied
gene
wildlooked
of
The
non-waxy
and
deficiency
intermediate
intermediate
rice,
wild-
amylose
by
12.8%.
no
higher
of
12-KI
rice.
mutants
to
also
the
the
low
purple
from
char-
mutants
the
non-waxy
distinct
percentage
the
The
reddish
were
had
starch
with
endosperm,
and
stained
to
They
or
to
maize
wx
endosperm
brown.
2).
endosperm
the
blue-black
normal
little
which
due
The
many
with
relative
wild-type
the
turned
physical
(Table
in
and
Although
were
tion,
the
amylose
MNU,
but
type,
mutation
translucent,
counterpart
starch
(du)
synthase ‡V.5
of
the
containing
content
dull
than
type
dull
amylose
the
starch
rice
snow
rice
lower
with
obtained
(wx)
and
content
soluble
cells
content
were
waxy
ratio
profoundly
egg
structure
contrast,
The
affect
amylose
into
by
type.
to
rice.
fertilized
amylose,
acterized
amylopectin.
modified
amylopectin
were
80%
is known
was
a
In
involved
all
of
correlation
addition
in
starch
those
the
lower-
have
and frequencies
to
been
of endosperm
identified in rice.6
) The gelatinization
pattern of endosperm
starch in urea
solutions of varying concentrations
was significantly
different among waxy, dull and non-waxy rice. Although the
initiation
of starch gelatinization
was almost the same in
all mutants, the termination
of gelatinization
was apparently distinct among them and increased in positively correlated with the amylose
content.
The swelling of endosperm starch from both waxy and non-waxy rice exhibited a biphasic response with increases in urea concentration. However, the extent of swelling at a given concentration of urea was more significant
in waxy starch than
in non-waxy starch. The results of gelatinization
pattern in
urea solution showed that the amylose content does not
affect
the initiation
of gelatinization,
but influences
greatly the swelling of gel, indicating
that amylose content affects the swelling power of gel and the termination
of gelatinization.
There was little difference in the initiation temperature
of thermal
gelatinization
of endosperm
starch among
waxy, dull and non-waxy
rice, although
it was slightly
lower in waxy mutants than in non-waxy rice. However,
the termination
temperature
of gelatinization
of endosperm
starch remarkably
differed. With increasing
amylose content, the termination
temperature
of gelatinization
shifted
to high temperature
as found in urea gelatinization.
As
mentioned
above, amylopectin
fine structure was not almutations
induced by MNU treatment
in rice.
Starch Mutants
tered by these mutations. These results suggest that amylopectin, the major component of starch, plays an important role for the initiation of gelatinization, whereas amylose contributes to the termination temperature.
Most of the waxy mutants induced by the MNU treatment were deficient in GBSSI, indicating that the lack of
amylose in the starches of waxy mutants is most likely
due to the deficiency in GBSSI. Some of the waxy mutants, the missense mutation of GBSSI gene, exhibited
normal levels of GBSSI. Expression level of GBSSI in
waxy or dull mutants with low amylose content correlated
well with amylose content, implying that GBSSI is responsible for amylose synthesis. However, the presence of
various mutant genes that lowered amylose content suggests that there is a complex mechanism for the genetic
regulation of amylose synthesis in rice. Recently, Issiki et
al .7)reported that dull mutations are involved in the splicing of mRNA of the Wxbgene.
The amylose content of endosperm starch greatly affects the cooking and eating qualities of milled rice, low
amylose varieties being more palatable than high amylose
ones, especially in japonica rice. Low amylose mutants
obtained with MNU treatment would be useful in the imppovement of palatability of rice.
Mutants affecting the biosynthesis of amylopectin.
Amylopectin is a highly branched molecule with a-1,6glucosidic bond and makes up about 80% of endosperm
starch in rice. The enzymatic mechanism of amylopectin
synthesis can be considered to involve four processes: initiation, chain elongation, branching and debranching.
These processes are catalyzed by 1) ADP-glucose pyrophosphorylase, 2) starch synthase, 3) starch branching enzyme and 4) starch debranching enzyme, respectively.
Various kinds of mutants for amylopectin biosynthesis
were recently found in maize, pea and Chlamydomonas.
We have also isolated various kinds of MNU-induced rice
mutants with modified amylopectin.
Three isoforms of branching enzyme-BET, BEITa and
BEIIb were observed in rice endosperm.8)9)Although the
amylose-extender (ae) mutant which is deficient in BEIIb,
were obtained by MNU treatment,10-12)
we recently isolated
mutants for the other two isoforms of BE, i.e., a mutant
lacking in BET activity and a mutant with a remarkable
decrease in BEIIa activity (Fig. 1). All of the BE mutants
were controlled by respective single genes. Genetic analysis using respective cDNA probes indicated that they are
mutants of genes encoding the BET and BEIIa, respectively, and tentatively designated sbe 1 and sbe 2, respectively.
The grains of the BEIIb-deficient ae mutant were
markedly smaller than those of the wild-type, and the dry
weight was 65% of the wild-type. In addition, the grains
were usually floury in appearance. Interestingly, the endosperm of BET mutant exhibited the normal phenotype
and contained an amount of starch comparable with the
wild-type. However, the mutation apparently altered the
fine structure of amylopectin. The same tendency was observed in the BEIIa mutation.
The a-1,4 chains of amylopectin consist of A chains
that carry no additional chains, B chains that carry A
in Rice
227
chains or other B chains, and a C chain that includes the
reducing terminus.13) Hizukuri14) proposed a cluster model
for amylopectin. In this model, A and B, chains form a
single cluster, while B2 and B3 chains extend to two and
three clusters, respectively. Hanashiro et al.)S) proposed
that, in amylopectin, chains of DP 12, 13 s DP s 24, 25
DP s 36 and DP 37 correspond to A chains, B,
chains, B2 chains, and B3 and longer chains, respectively.
The profiles of chain length distribution were significantly different among BE mutations (Fig. 2). BEIIb (ae )
mutation decreased specifically the short chains with DP
less than 17, suggesting that the BEIIb contributes to the
synthesis of A chains of amylopectin. In contrast, the
amylopectin of BET deficient mutant (sbel ) was characterized by a significant decrease in long chains with DP
longer than 38 and short chains with DP 12 to 23, and a
increase in short chains with shorter than DP 11 and in
intermediate chains with DP 24 to 35. This suggests that
BEI specifically synthesizes B, chains as well as B3
chains. However, notable alteration on the profile of chain
length distribution was not observed in BEIIa (sbe2) mutation. The structure of amylopectin is characterized by
the fact that a unit structure with a constant size throughout the plant kingdom called cluster is tandem linked,16" )
and the distinct structure may be referred to as "tandemcluster structure. " It has been documented that the individual cluster has the amorphous region and the crystalline region, and branches are distributed in the both regions.)8"9) In the rice BE mutants, the loss of BE activity
leads to poor efficiency in producing unit-chain (branches)
of amylopectin. Based on these observations, it is reasonable to assume that BEs should produce three different
types of branches in terms of these contributions to the
amylopectin structure; those formed in the amorphous region and the crystalline region of the cluster, and those
linking the clusters.20)
The effects of urea concentrations from 0 to 9 M on the
gelatinization of starch granules were distinctively observed in three kinds of BE mutants. The starch from mature endosperm of the wild-type started to gelatinize between 3 and 4 M urea solution. By contrast, endosperm
starch granules from the ae mutant hardly gelatinized in
urea solution up to 7 M, but began to gelatinize at 8 M.
The starch granules from the amylose-free ae/wx double
mutant, which consisted exclusively of ae-type amylopectin, also gelatinized in 8 M urea. These results indicate
that the starting concentration of urea for gelatinization of
endosperm starch granules depends on the structure of the
amylopectin rather than on the level or structure of amylose. On the contrary, the sbel mutant starch exhibited a
slightly lower concentration of urea for onset gelatinization and a higher swelling mode as compared with the
wild-type starch. The endosperm starch from the sbe2
mutant exhibited little difference with the wild-type on the
concentration of urea for onset gelatinization.
Each BE mutation showed distinct effect on the thermal
properties of endosperm starch (Table 3). While BEIIbdeficient ae starch exhibited extremely higher onset gelatinization temperature, the sbel starch was characterized
by a lower onset temperature for thermo-gelatinization
and a significantly lower enthalpy. These results indicate
228
Table
J. Appl. Glycosci.,
3.
Thermal properties of starch from endosperms
three kinds of rice BE mutants as measured
DSC.
Vol. 50, No. 2 (2003)
of
by
Fig. 3.
Effect of sugary mutations on chain length
amylopectin in rice endosperm.
distribution
of
The figures compare the chain profiles of total polyglucans
between sugary I and sugary 2 mutants and their parent cultivars,
Taichung 65 and Kinmaze, respectively.
Fig. 1.
SDS-PAGE and Native-PAGE/activity staining analyses
of enzymes involved in starch biosynthesis in three kinds
of BE mutants of rice.
A) SDS-PAGE of enzymes in mature rice seed. B) NativePAGE/activity staining of BEs in developing rice seed.
Fig. 4.
SDS-PAGE and native-PAGE/activity staining analyses of
enzymes involving in starch biosynthesis in developing
seeds of _floury 2 mutant in rice.
A) SDS-PAGE of enzymes in mature seed. B) Native-PAGE/activity staining of BEs in developing seed.
Fig. 5.
Effect of floury 2 mutation on chain length distribution of
amylopectin in rice endosperm.
The figures compare the chain length profiles of amylopectins
between the mutant (EM36) and its parent cultivars Kinmaze.
that the genetic modification of amylopectin fine structure
is responsible for changes in physicochemical properties
of starch. The BEIIa mutant starch had the highest enthalpy, though no significant alteration in the chain length
distribution of amylopectin was found (Fig. 2), suggesting
that a-1,6 branches produced by BElla also affect the
ultra-structure of amylopectin.
Two kinds of debranching enzymes-isoamylase
and
Fig. 2.
Effect of BE mutations on chain length distribution of
amylopectin in rice endosperm.
The figures show differences in chain length profiles of amylopectins between the respective mutants and their parent cultivars,
Taichung 65 (for sbel mutant) and Kinmaze (for ae and sbe2 mutants) (japonica -type rice).
pullulanase are found in endosperm cell of rice.21)wo
kinds of non-allelic sugary mutant which are named as
sugary I (sugl )22)and sugary 2 (sug2), respectively, have
been identified in rice. Endosperm starch of sugl mutants
is characterized by the higher content of the water soluble
polysaccharide phytoglycogen having less amount of amylopectin.2'.24' Kubo et a1.2" reported that sugl mutation in
rice is lesion of the isoamylase gene and it causes the remarkable reduction of pullulanase activity. Preliminary
experiment indicated that sug2 mutation also significantly
Starch
Fig. 6.
Schematic
representation
of the metabolic
pathways
Mutants
and putative
in Rice
sites of mutations
229
in starch biosynthesis
of rice endosperm.
1, invertase;
2, hexokinase;
3, hexose-6-phosphatase;
4, P-hexoseisomerase;
5, cytosolic
P-glucomutase;
6, sucrose
synthase;
7, UDPGIcPPase;
8, cytosolic ADPglucose
pyrophosphorylase;
9, plastidial P-glucomutase;
10, plastidial ADPglucose
pyrophosphorylase;
11, plastidial starch phosphorylase.
ADPG, ADPglucose;
UDPG, UDPglucose.
reduced the expression level of pullulanase in endosperm.
Figure 3 shows the effects of sugl and sug2 mutations
on the chain length distribution of amylopectin. The sug]
mutation accumulated phytoglycogen instead of starch. In
phytoglycogen, the number of A chains dramatically increased, while long B chains with less than DP 37 remarkably decreased or were almost absent, which resulted
in the disappearance of the cluster structure .21)The sug2
mutation accumulated a significant amount of the water
soluble polysaccharides. Both sugary mutations increased
short chains and decreased long chains. So far, five strains
of sug2 mutants were isolated by independent MNU treatment. The expression level of pullulanase was decreased
in all sug2 mutants. Mutants lacking in this enzyme has
not been isolated. Genetic analysis suggested that sug2
gene is not in chromosome 4 where a gene encoding pullulanase is located26) implying that sug2 gene might participate in the regulation of pullulanase, but not encode
this enzyme.
An interesting mutation floury 2 (flo2) was found in
rice. Western blot analysis showed that the BEI level was
greatly reduced to almost 10% of the wild-type in the mature seeds of the flo2 mutants.2" Interestingly, the production of BEIIb, SSSI, GBSS, PUL and starch phosphorylase was also reduced (Fig. 4). We also found that the expression levels of BEIIb, GBSS and PUL in the flo2 mutant corresponded to approximately 55, 80 and 35%, respectively, of those in the wild-type (data not shown).
The flo2 locus and the genes encoding BEI, BEIlb and
GBSS have been mapped on different chromosomes.
Therefore, the wild type Flo2 gene probably encodes a
regulatory protein that modulates the expression of the
genes involved in starch synthesis, particularly BEI.
Since the 11 flo2 mutants exhibited reduced levels of
BEIIb, they were expected to contain a higher proportion
of apparent amylose content in starch than that of the
wild-type. On the contrary, these 11 mutants had a significantly decreased amylose content (9-11%), as compared with the wild-type (15%). The reduction of amylose
content in endosperm starch of flo2 mutant is probably
caused by the significant reduction of the expression level
of GBSSI.
The flo2 mutation also altered the fine structure of
amylopectin (Fig. 5). The flo2 amylopectin was characterized by the significant decrease in long chains with longer
than DP 36 and short chains with DP 9-20, and the increases markedly in short chains with shorter than DP 8
and slightly in intermediate chains with DP 21-35. The
profile of chain length distribution in flo2 amylopectin
was similar to that of BEI mutant. The mode of gelatinization of endosperm starch by urea was also modified by
the flo2 mutation. The flo2 starch was resistant to urea
solution relative to that of the wild-type starch. The swelling of flo2 starch was lower than that of the wild-type,
although the amylose content was significantly lower than
that of the wild-type.
In spite of the low level of BEI gene expression in the
mature endosperm of the flo2 mutants, the BEI gene was
equally expressed in the leaves of the wild-type and flo2
mutants. These results imply that wild type Flog gene
may co-regulate the expression of some of the genes participating in starch synthesis possibly in a developing specific manner of the seed.
In addition to the mutants modifying the starch properties, some mutants involved in the biosynthesis of ADPglucose were isolated in rice as well as in maize 21,21,or
pea."" Two types of the mutant, i.e., shrunken I (shrl )
and shrunken 2 (shr2) were isolated in rice.22)These mutants were characterized by the reduced amount of starch
and the elevated amount of sugars. Multiple allelic mutants were observed in both types. Preliminary experiments showed that both mutants exhibited less than 20%
of ADP-glucose pyrophosphorylase activity of wild-type.
Western blot analysis using antibodies for maize ADPglucose pyrophophorylase large subunit and small subunit
showed that the endosperm of shr2 mutant lacked the
small subunit, suggesting that the shr2 mutation occurs in
a gene for ADP-glucose pyrophophorylase small subunit.
J. Appl. Glycosci., Vol. 50, No. 2 (2003)
230
By contrast, shrl mutants showed equal expression of
both of ADP-glucose pyrophophorylase large subunit and
small subunit. The detail mechanisms for the shrunken
mutations await further investigation.
Many kinds of mutant genes involving in the starch
biosynthesis have been identified in rice (Fig. 6) as well
as in maize, pea or Chlamydomonas .31' There are many
questions to be resolved for the biosynthesis of starch, although the recent progress in understanding its genetic
regulation is encouraging. The analysis of mutants affecting the starch-synthesizing enzyme will provide clues to
clarify the in vivo function of the corresponding enzymes.20' These clues will be useful in the breeding of rice
varieties having novel starch properties.
347
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of
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16)
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ture
in
the
sources.
17)
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S.
structure
of
Marcel
Dekker,
Bertoft
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and
A.M.
distribution
revealed
by
high
Carbohydr.
417-420
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