Isolation and Characterization of Starch Mutants in Rice
(Received September 15, 2002)
Hikaru Satoh,1,* Aiko Nishi,1 Naoko Fujita,2,3 Akiko Kubo,2,3 Yasunori 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 affecting the amylopectin structure. The amylose content did not affect the initiation of gelatinization, but influenced greatly the swelling of gel, indicating that amylose content affects the swelling power of gel and the ter-mination 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 endosperm starch. The sug2 mutation lowering the expression level of PUL increased shorter chains of amylopectin and accumulated a significant amount of the water-soluble polysaccharides as well as ISA (sugl ) mutation. 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 biosynthesis 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 im-
portant goals in rice breeding because starch properties greatly influence the eating and cooking qualities of rice in addition to its processing method for industrial use. For
the purpose, the genetic resources for improving starch
properties must be generated, collected, characterized and evaluated. In maize, various kinds of mutants for polysac-charides reserves in the grain such as waxy, sugary, shrunken and amylose-extender have been identified. These mutants have greatly contributed to the improve-ment of starch quality of maize, and thus expanded the use of maize not only as food but also for important ma-terials for food industry. In addition to the importance of improving grain quality, maize mutants have provided valuable information on gene action in metabolic regula-tion 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 N-methyl-N nitorosourea (MNU)1) and obtained several thousands mutations for embryo and endosperm proper-ties2'3' as numerous as those in maize. Using these NMU-induced mutants, the genetics and the effects of specific
genes on qualitative and quantitative changes in carbohy-drates 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 be-ing white core mutant. This might be due to the differ-ence 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 fer-tilized egg cell treatment is characterized by high muta-tion 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 point-mutations, not deletion type as found by gamma irradia-tion 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
20% amylose and 80% amylopectin. The ratio of amylose
to amylopectin is known to affect profoundly the physical
properties of cooked rice.
After treatment of fertilized egg cells with MNU, many
mutants with modified amylose content but with normal
amylopectin fine structure were obtained (Table 2). They
were classified into waxy (wx) type containing little or no
amount of amylose, and dull (du) type, which were char-
acterized by lower amylose content relative to the wild-
type. In contrast, the dull mutation in maize had higher
amylose content than the wild-type due to the deficiency
in soluble starch synthase ‡V.5 The endosperm of wild-
type rice was translucent, and the wx mutants looked
snow white. Although the endosperm starch of non-waxy
rice counterpart turned blue-black with 12-KI solution, wx
starch was stained light brown. The low amylose (dull)
mutants had hazy white endosperm, the intermediate be-
tween waxy rice and non-waxy rice. The dull mutant
starches were stained reddish purple by the iodine solu-
tion, which were distinct from non-waxy or waxy rice.
Amylose percentage in dull mutants varied among mutant
lines ranging from 3.2% to 12.8%. The phenotypes of all
the dull mutants were also intermediate between those of
the non-waxy and waxy rice, and there was a correlation
between phenotype and amylose content. In addition to
the wx locus, five other gene loci involved in the lower-
ing of amylose contents of endosperm starch have been
identified in rice.6
) The gelatinization pattern of endosperm starch in urea
solutions of varying concentrations was significantly dif-
ferent 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 appar-
ently distinct among them and increased in positively cor-
related with the amylose content. The swelling of en-
dosperm starch from both waxy and non-waxy rice exhib-
ited a biphasic response with increases in urea concentra-
tion. However, the extent of swelling at a given concen-
tration 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 con-tent 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 con-
tent, the termination temperature of gelatinization shifted
to high temperature as found in urea gelatinization. As
mentioned above, amylopectin fine structure was not al-
Table 1. Spectra and frequencies of endosperm mutations induced by MNU treatment in rice.
227Starch Mutants in Rice
tered by these mutations. These results suggest that amy-lopectin, the major component of starch, plays an impor-tant role for the initiation of gelatinization, whereas amy-lose contributes to the termination temperature.
Most of the waxy mutants induced by the MNU treat-ment 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 mu-tants, 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 re-sponsible for amylose synthesis. However, the presence of various mutant genes that lowered amylose content sug-
gests 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 splic-ing of mRNA of the Wxb gene. The amylose content of endosperm starch greatly af-
fects 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 im-
ppovement of palatability of rice.
Mutants affecting the biosynthesis of amylopectin. Amylopectin is a highly branched molecule with a-1,6-
glucosidic bond and makes up about 80% of endosperm starch in rice. The enzymatic mechanism of amylopectin synthesis can be considered to involve four processes: ini-tiation, chain elongation, branching and debranching. These processes are catalyzed by 1) ADP-glucose pyro-
phosphorylase, 2) starch synthase, 3) starch branching en-zyme 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 analy-sis using respective cDNA probes indicated that they are mutants of genes encoding the BET and BEIIa, respec-tively, and tentatively designated sbe 1 and sbe 2, respec-tively. 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 en-dosperm 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 ob-served 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
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 signifi-
cantly 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 character-ized 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) mu-tation. The structure of amylopectin is characterized by the fact that a unit structure with a constant size through-out the plant kingdom called cluster is tandem linked,16" ) and the distinct structure may be referred to as "tandem-cluster structure. " It has been documented that the indi-
vidual cluster has the amorphous region and the crystal-line region, and branches are distributed in the both re-
gions.)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 reason-able 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 re-
gion 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 ob-served in three kinds of BE mutants. The starch from ma-ture endosperm of the wild-type started to gelatinize be-tween 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 amylopec-tin, 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 amy-lose. On the contrary, the sbel mutant starch exhibited a
slightly lower concentration of urea for onset gelatiniza-tion 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 BEIIb-deficient ae starch exhibited extremely higher onset ge-latinization temperature, the sbel starch was characterized by a lower onset temperature for thermo-gelatinization and a significantly lower enthalpy. These results indicate
228 J. Appl. Glycosci., Vol. 50, No. 2 (2003)
Table 3. Thermal properties of starch from endosperms of
three kinds of rice BE mutants as measured by
DSC.
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) Native-PAGE/activity staining of BEs in developing rice seed.
Fig. 2. Effect of BE mutations on chain length distribution of amylopectin in rice endosperm.
The figures show differences in chain length profiles of amy-lopectins between the respective mutants and their parent cultivars, Taichung 65 (for sbel mutant) and Kinmaze (for ae and sbe2 mu-tants) (japonica -type rice).
Fig. 3. Effect of sugary mutations on chain length distribution of
amylopectin in rice endosperm.
The figures compare the chain profiles of total polyglucans be-
tween sugary I and sugary 2 mutants and their parent cultivars,
Taichung 65 and Kinmaze, respectively.
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/ac-tivity 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 en-thalpy, 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
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 amy-lopectin.2'.24' Kubo et a1.2" reported that sugl mutation in rice is lesion of the isoamylase gene and it causes the re-markable reduction of pullulanase activity. Preliminary experiment indicated that sug2 mutation also significantly
229Starch Mutants in Rice
Fig. 6. Schematic representation of the metabolic pathways and putative sites of mutations in starch biosynthesis of rice endosperm.
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 in-creased, while long B chains with less than DP 37 re-markably 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 treat-ment. 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 pul-lulanase is located26) implying that sug2 gene might par-ticipate 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 ma-ture seeds of the flo2 mutants.2" Interestingly, the produc-tion of BEIIb, SSSI, GBSS, PUL and starch phosphory-lase was also reduced (Fig. 4). We also found that the ex-
pression levels of BEIIb, GBSS and PUL in the flo2 mu-tant corresponded to approximately 55, 80 and 35%, re-spectively, 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 sig-nificantly decreased amylose content (9-11%), as com-
pared 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 character-ized by the significant decrease in long chains with longer
than DP 36 and short chains with DP 9-20, and the in-creases 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 gelatini-zation 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 swel-ling 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 par-ticipating in starch synthesis possibly in a developing spe-cific manner of the seed.
In addition to the mutants modifying the starch proper-ties, some mutants involved in the biosynthesis of ADP-
glucose 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 mu-tants were characterized by the reduced amount of starch and the elevated amount of sugars. Multiple allelic mu-tants were observed in both types. Preliminary experi-ments showed that both mutants exhibited less than 20% of ADP-glucose pyrophosphorylase activity of wild-type. Western blot analysis using antibodies for maize ADP-
glucose 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.
230 J. Appl. Glycosci., Vol. 50, No. 2 (2003)
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, al-though the recent progress in understanding its genetic
regulation is encouraging. The analysis of mutants affect-ing the starch-synthesizing enzyme will provide clues to
clarify the in vivo function of the corresponding en-zymes.20' These clues will be useful in the breeding of rice varieties having novel starch properties.
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