•Protein–protein complex formation during starch biosynthesis.•Differences in starch biosynthetic protein complexes among cereals.•Starch biosynthetic protein complex formation in ...mutants.•Involvements of enzyme interactions in the initiation of starch biosynthesis.•Future perspectives and methods for analyzing functions of protein complexes.
Starch accounts for the majority of edible carbohydrate resources generated through photosynthesis. Amylopectin is the major component of starch and is one of highest-molecular-weight biopolymers. Rapid and systematic synthesis of frequently branched hydro-insoluble amylopectin and efficient accumulation into amyloplasts of cereal endosperm is crucial. The functions of multiple starch biosynthetic enzymes, including elongation, branching, and debranching enzymes, must be temporally and spatially coordinated. Accordingly, direct evidence of protein–protein interactions of starch biosynthetic enzymes were first discovered in developing wheat endosperm in 2004, and they have since been shown in the developing seeds of other cereals. This review article describes structural characteristics of starches as well as similarities and differences in protein complex formation among different plant species and among mutant plants that are deficient in specific starch biosynthetic enzymes. In addition, evidence for protein complexes that are involved in the initiation stages of starch biosynthesis is summarized. Finally, we discuss the significance of protein complexes and describe new methods that may elucidate the mechanisms and roles of starch biosynthetic enzyme complexes.
Key message
Introduction of higher SSIIa activity to mild-type
isa1
mutant by crossing results in restoration of crystallinity, starch granule structure, and production of plump seeds.
Isoamylase 1 ...(ISA1) removes improper α-1, 6 glycosidic branches of amylopectin generated by starch branching enzymes and is essential for the formation of proper amylopectin structure. Rice
isa1
(
sug-1
) mutants in japonica cultivar with less-active starch synthase IIa (SSIIa) and low granule-bound SSI (GBSSI) expression display wrinkled seed phenotype by accumulating water-soluble phytoglycogen instead of insoluble amylopectin. Expression of active SSIIa in transgenic rice produced with a severe-type
isa1
mutant accumulated some insoluble glucan with weak B-type crystallinity at the periphery of seeds but their seeds remained wrinkled. To see whether introduction of high levels of SSIIa and/or GBSSI can restore the grain filling of the mild-type
sug-1
mutant (EM653), new rice lines (
SS2a gbss1
L
isa1
,
ss2a
L
GBSS1 isa1
, and
SS2a GBSS1 isa1
) were generated by crossing japonica
isa1
mutant (
ss2a
L
gbss1
L
isa1
) with wild type indica rice (
SS2a GBSS1 ISA1
). The results showed that
SS2a gbss1
L
isa1
and
SS2a GBSS1 isa1
lines generated chalky plump seeds accumulating insoluble amylopectin-like glucans with an increase in DP 13–35, while
ss2a
L
GBSS1 isa1
generated wrinkly seeds and accumulated soluble glucans enriched with DP < 13. Scanning electron microscopic observation of cross-section of the seeds showed that
SS2a gbss1
L
isa1
and
SS2a GBSS1 isa1
produced wild type-like polygonal starch granules. These starches showed the A-type crystallinity comparable to the wild type, while the japonica
isa1
mutant and the transgenic rice do not show any or little crystallinity, respectively. These results indicate that introduction of higher SSIIa activity can mostly complements the mild-type
sug-1
phenotype.
Background
Cereals high in resistant starch (RS) are gaining popularity, as their intake is thought to help manage diabetes and prediabetes. Number of patients suffering from diabetes is also ...increasing in Asian countries where people consume rice as a staple food, hence generation of practically growable high RS rice line has been anticipated. It is known that suppression of starch branching enzyme (BE) IIb increases RS content in cereals. To further increase RS content and for more practical use, we generated a non-transgenic
be1 be2b
double mutant rice (
Oryza sativa
) line, which completely lacked both proteins, by crossing a
be1
mutant with a
be2b
mutant.
Results
The
be1 be2b
mutant showed a decrease in intermediate amylopectin chains and an increase in long amylopectin chains compared with
be2b
. The amylose content of
be1 be2b
mutant (51.7%) was the highest among all pre-existing non-transgenic rice lines. To understand the effects of chewing cooked rice and cooking rice flour on RS content, RS content of mashed and un-mashed cooked rice as well as raw and gelatinized rice flour were measured using
be1 be2b
and its parent mutant lines. The RS contents of mashed cooked rice and raw rice flour of
be1 be2b
mutant (28.4% and 35.1%, respectively) were 3-fold higher than those of
be2b
mutant. Gel-filtration analyses of starch treated with digestive enzymes showed that the RS in
be1 be2b
mutant was composed of the degradation products of amylose and long amylopectin chains. Seed weight of
be1 be2b
mutant was approximately 60% of the wild type and rather heavier than that of
be2b
mutant.
Conclusions
The endosperm starch in
be1 be2b
double mutant rice were enriched with long amylopectin chains. This led to a great increase in RS content in cooked rice grains and rice flour in
be1 be2b
compared with
be2b
single mutant.
be1 be2b
generated in this study must serve as a good material for an ultra-high RS rice cultivar.
Amylopectin is a highly branched, organized cluster of glucose polymers, and the major component of rice starch. Synthesis of amylopectin requires fine co-ordination between elongation of glucose ...polymers by soluble starch synthases (SSs), generation of branches by branching enzymes (BEs), and removal of misplaced branches by debranching enzymes (DBEs). Among the various isozymes having a role in amylopectin biosynthesis, limited numbers of SS and BE isozymes have been demonstrated to interact via protein–protein interactions in maize and wheat amyloplasts. This study investigated whether protein–protein interactions are also found in rice endosperm, as well as exploring differences between species. Gel permeation chromatography of developing rice endosperm extracts revealed that all 10 starch biosynthetic enzymes analysed were present at larger molecular weights than their respective monomeric sizes. SSIIa, SSIIIa, SSIVb, BEI, BEIIb, and PUL co-eluted at mass sizes >700 kDa, and SSI, SSIIa, BEIIb, ISA1, PUL, and Pho1 co-eluted at 200–400 kDa. Zymogram analyses showed that SSI, SSIIIa, BEI, BEIIa, BEIIb, ISA1, PUL, and Pho1 eluted in high molecular weight fractions were active. Comprehensive co-immunoprecipitation analyses revealed associations of SSs–BEs, and, among BE isozymes, BEIIa–Pho1, and pullulanase-type DBE–BEI interactions. Blue-native-PAGE zymogram analyses confirmed the glucan-synthesizing activity of protein complexes. These results suggest that some rice starch biosynthetic isozymes are physically associated with each other and form active protein complexes. Detailed analyses of these complexes will shed light on the mechanisms controlling the unique branch and cluster structure of amylopectin, and the physicochemical properties of starch.
Key message
Mutation of the
BEIIb
gene in an
isa1
mutant background mitigates the negative effect of the
ISA1
mutation on grain filling, and facilitates recovery of amyloplast formation in rice ...endosperm.
In this study, the effect of branching enzyme IIb and isoamylase 1 deficiency on starch properties was demonstrated using high resistant starch rice lines, Chikushi-kona 85 and EM129. Both lines harbored a mutation in the
BEIIb
and
ISA1
genes and showed no BEIIb and ISA1 activity, implying that both lines are
beIIb isa1
double mutants. The amylopectin long chain and apparent amylose content of both mutant lines were higher than those of the wild-type. While both mutants contained loosely packed, round starch grains, a trait specific to
beIIb
mutants, they also showed collapsed starch grains at the center of the endosperm, a property specific to
isa1
mutants. Furthermore,
beIIb isa1
double mutant F
2
lines derived from a cross between Chikushi-kona 85 and Nishihomare (wild-type cultivar) showed significantly heavier seed weight than the
beIIb
and
isa1
single mutant lines. These results suggest that co-occurrence of
beIIb
and
isa1
mutant alleles in a single genetic background mitigates the negative effect of the
isa1
allele on grain filling, and contributes to recovery of the amyloplast formation defect in the
isa1
single mutant.
The lengths of amylopectin-branched chains are precise and influence the physicochemical properties of starch, which determine starch functionality. Three major isozymes of starch synthases (SSs), ...SSI, SSII(a), and SSIII(a), are primarily responsible for amylopectin chain elongation in the storage tissues of plants. To date, the majority of reported rice mutants were generated using japonica cultivars, which have almost inactive SSIIa. Although three SSs share some overlapping chain length preferences, whether they complement each other remains unknown due to the absence of suitable genetic combinations of materials. In this study, rice
ss1
/
SS2a
/
SS3a
and
SS1
/
SS2a
/
ss3a
were newly generated, and the chain length distribution patterns of all the possible combinations of presence and absence of SSI, SSIIa, and SSIIIa activities were compared. This study demonstrated that SSIIa can complement most SSI functions that use glucan chains with DP 6–7 to generate DP 8–12 chains but cannot fully compensate for the elongation of DP 16–19 chains. This suggests that SSIIa preferentially elongates outer but not inner chains of amylopectin. In addition, the results revealed that neither SSI nor SSIIIa compensate for SSIIa. Neither SSI nor SSIIa compensate for elongation of DP >30 by SSIIIa. SSIIa could not resolve the pleiotropic increase of SSI caused by the absence of SSIIIa; instead, SSIIa further elongated those branches elongated by SSI. These results revealed compensatory differences among three major SS isozymes responsible for lengths of amylopectin branches.
Rice endosperm accumulates large amounts of photosynthetic products as insoluble starch within amyloplasts by properly arranging structured, highly branched, large amylopectin molecules, thus ...avoiding osmotic imbalance. The amount and characteristics of starch directly influence the yield and quality of rice grains, which in turn influence their application and market value. Therefore, understanding how various allelic combinations of starch biosynthetic genes, with different expression levels, affect starch properties is important for the identification of targets for breeding new rice cultivars. Research over the past few decades has revealed the spatiotemporal expression patterns and allelic variants of starch biosynthetic genes, and enhanced our understanding of the specific roles and compensatory functions of individual isozymes of starch biosynthetic enzymes through biochemical analyses of purified enzymes and characterization of
japonica
rice mutants lacking these enzymes. Furthermore, it has been shown that starch biosynthetic enzymes can mutually and synergistically increase their activities by forming protein complexes. This review focuses on the more recent discoveries made in the last several years. Generation of single and double mutants and/or high-level expression of specific starch synthases (SSs) allowed us to better understand how the starch granule morphology is determined; how the complete absence of SSIIa affects starch structure; why the rice endosperm stores insoluble starch rather than soluble phytoglycogen; how to elevate amylose and resistant starch (RS) content to improve health benefits; and how SS isozymes mutually complement their activities. The introduction of active-type
SSIIa
and/or high-expression type
GBSSI
into
ss3a ss4b
,
isa1
,
be2b
, and
ss3a be2b japonica
rice mutants, with unique starch properties, and analyses of their starch properties are summarized in this review. High-level accumulation of RS is often accompanied by a reduction in grain yield as a trade-off. Backcrossing rice mutants with a high-yielding elite rice cultivar enabled the improvement of agricultural traits, while maintaining high RS levels. Designing starch structures for additional values, breeding and cultivating to increase yield will enable the development of a new type of rice starch that can be used in a wide variety of applications, and that can contribute to food and agricultural industries in the near future.
Starch synthase (SS) IIIa has the second highest activity of the total soluble SS activity in developing rice endosperm. Branching enzyme (BE) IIb is the major BE isozyme, and is strongly expressed ...in developing rice endosperm. A mutant (ss3a/be2b) was generated from wild-type japonica rice which lacks SSIIa activity. The seed weight of ss3a/be2b was 74–94% of that of the wild type, whereas the be2b seed weight was 59–73% of that of the wild type. There were significantly fewer amylopectin short chains degree of polymerization (DP) ≤13 in ss3a/be2b compared with the wild type. In contrast, the amount of long chains (DP ≥25) connecting clusters of amylopectin in ss3a/be2b was higher than in the wild type and lower than in be2b. The apparent amylose content of ss3a/be2b was 45%, which was >1.5 times greater than that of either ss3a or be2b. Both SSIIIa and BEIIb deficiencies led to higher activity of ADP-glucose pyrophosphorylase (AGPase) and granule-bound starch synthase I (GBSSI), which partly explains the high amylose content in the ss3a/be2b endosperm. The percentage apparent amylose content of ss3a and ss3a/be2b at 10 days after flowering (DAF) was higher than that of the wild type and be2b. At 20 DAF, amylopectin biosynthesis in be2b and ss3a/be2b was not observed, whereas amylose biosynthesis in these lines was accelerated at 30 DAF. These data suggest that the high amylose content in the ss3a/be2b mutant results from higher amylose biosynthesis at two stages, up to 20 DAF and from 30 DAF to maturity.
The gelatinization temperature of endosperm starch in most japonica rice cultivars is significantly lower than that in most indica rice cultivars. This is because three single nucleotide ...polymorphisms in the
(
)
gene in japonica rice cultivars (
) significantly reduce SSIIa activity, resulting in an increase in amylopectin short chains with degree of polymerization (DP) ≤ 12 compared to indica rice cultivars (
). SSIIa forms a trimeric complex with SSI and starch branching enzyme (BE) IIb in maize and japonica rice, which is likely important for the biosynthesis of short and intermediate amylopectin chains (DP ≤ 24) within the amylopectin cluster. It was unknown whether the complete absence of SSIIa further increases amylopectin short chains and reduces gelatinization temperature and/or forms altered protein complexes due to the lack of a suitable mutant. Here, we identify the SSIIa-deficient mutant rice line
(
) from a screen of ca. 1,500 plants of the rice cultivar Kinmaze (japonica) that were subjected to
-methyl-
-nitrosourea mutagenesis. The
gene in
was mutated at the boundary between intron 5 and exon 6, which generated a guanine to adenine mutation and resulted in deletion of exon 6 in the mRNA transcript. SSIIa activity and SSIIa protein in developing endosperm of
were not detected by native-PAGE/SS activity staining and native-PAGE/immunoblotting, respectively. SSIIa protein was completely absent in mature seeds. Gel filtration chromatography of soluble protein extracted from developing seeds showed that the SSI elution pattern in
was altered and more SSI was eluted around 300 kDa, which corresponds with the molecular weight of trimeric complexes in wild type. The apparent amylose content of
rice grains was higher than that in its parent Kinmaze.
also had higher content of amylopectin short chains (DP ≤ 12) than Kinmaze, which reduced the gelatinization temperature of
starch by 5.6°C compared to Kinmaze. These results indicate that
starch will be suitable for making foods and food additives that easily gelatinize and slowly retrograde.
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