Dataset Information

Analyses of starch biosynthetic protein complexes and starch properties from developing mutant rice seeds with minimal starch synthase activities.

ABSTRACT: Starch is the major component of cereal grains and is composed of essentially linear amylose and highly branched amylopectin. The properties and composition of starch determine the use and value of grains and their products. Starch synthase (SS) I, SSIIa, and SSIIIa play central roles in amylopectin biosynthesis. These three SS isozymes also affect seed development, as complete loss of both SSI and SSIIIa under reduced SSIIa activity in rice lead to sterility, whereas presence of minimal SSI or SSIIIa activity is sufficient for generating fertile seeds. SSs, branching enzymes, and/or debranching enzymes form protein complexes in cereal. However, the relationship between starch properties and the formation of protein complexes remain largely unknown. To better understand this phenomenon, properties of starch and protein complex formation were analyzed using developing mutant rice seeds (ss1 L /ss2a L /ss3a) in which all three major SS activities were reduced.The SS activity of ss1 L /ss2a L /ss3a was 25%-30% that of the wild-type. However, the grain weight of ss1 L /ss2a L /ss3a was 89% of the wild-type, 55% of which was starch, showing considerable starch synthesis. The reduction of soluble SS activity in ss1 L /ss2a L /ss3a resulted in increased levels of ADP-glucose pyrophosphorylase and granule-bound starch synthase I, which are responsible for substrate synthesis and amylose synthesis, respectively. Together, these features led to an increase in apparent amylose content (34%) in ss1 L /ss2a L /ss3a compared with wild-type (20%). Gel filtration chromatography of the soluble proteins in ss1 L /ss2a L /ss3a showed that the majority of the starch biosynthetic enzymes maintained the similar elution patterns as wild-type, except that the amounts of high-molecular-weight SSI (>?300 kDa) were reduced and SSIIa of approximately 200-300 kDa were present instead of those >?440 kDa, which predominate in wild-type. Immuno-precipitation analyses suggested that the interaction between the starch biosynthetic enzymes maybe reduced or weaker than in wild-type.Although major SS isozymes were simultaneously reduced in ss1 L /ss2a L /ss3a rice, active protein complexes were formed with a slightly altered pattern, suggesting that the assembly of protein complexes may be complemented among the SS isozymes. In addition, ss1 L /ss2a L /ss3a maintained the ability to synthesize starch and accumulated less amylopectin and more amylose in starch.

SUBMITTER: Hayashi M

PROVIDER: S-EPMC5894220 | biostudies-other | 2018 Apr

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Analyses of starch biosynthetic protein complexes and starch properties from developing mutant rice seeds with minimal starch synthase activities.

Hayashi Mari M Crofts Naoko N Oitome Naoko F NF Fujita Naoko N

BMC plant biology 20180410 1

<h4>Background</h4>Starch is the major component of cereal grains and is composed of essentially linear amylose and highly branched amylopectin. The properties and composition of starch determine the use and value of grains and their products. Starch synthase (SS) I, SSIIa, and SSIIIa play central roles in amylopectin biosynthesis. These three SS isozymes also affect seed development, as complete loss of both SSI and SSIIIa under reduced SSIIa activity in rice lead to sterility, whereas presence ...[more]

PMID: 29636002

Similar Datasets

Project description:Amylopectin, the major component of starch, is synthesized by synergistic activity of multiple isozymes of starch synthases (SSs) and branching enzymes (BEs). The frequency and length of amylopectin branches determine the functionality of starch. In the rice endosperm, BEIIb generates short side chains of amylopectin and SSI elongates those branches, which can be further elongated by SSIIa. Absence of these enzymes greatly affects amylopectin structure. SSI, SSIIa, and BEIIb associate with each other and with other starch biosynthetic enzymes although SSIIa is low activity in japonica rice. The aim of the current study was to understand how the activity of starch biosynthetic enzyme complexes is compensated in the absence of SSI or BEIIb, and whether the compensatory effects are different in the absence of BEIIb or in the presence of inactive BEIIb. Interactions between starch biosynthetic enzymes were analyzed using one ss1 null mutant and two be2b japonica rice mutants (a mutant producing inactive BEIIb and a mutant that did not produce BEIIb). Soluble proteins extracted from the developing rice seeds were separated by gel filtration chromatography. In the absence of BEIIb activity, BEIIa was eluted in a broad molecular weight range (60-700 kDa). BEIIa in the wild-type was eluted with a mass below 300 kDa. Further, majority of inactive BEIIb co-eluted with SSI, SSIIa, and BEI, in a mass fraction over 700 kDa, whereas only small amounts of these isozymes were found in the wild-type. Compared with the be2b lines, the ss1 mutant showed subtle differences in protein profiles, but the amounts of SSIIa, SSIVb, and BEI in the over-700-kDa fraction were elevated. Immunoprecipitation revealed reduced association of SSIIa and BEIIb in the ss1 mutant, while the association of BEIIb with SSI, SSIIa, SSIVb, BEI, and BEIIa were more pronounced in the be2b mutant that produced inactive BEIIb enzyme. Mass spectrometry and western blotting revealed that SSI, SSIIa, SSIIIa, BEI, BEIIa, starch phosphorylase 1, and pullulanase were bound to the starch granules in the be2b mutants, but not in the wild-type and ss1 mutant. These results will aid the understanding of the mechanism of amylopectin biosynthesis.

Project description: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 Starch synthase (SS) IIa gene in japonica rice cultivars (SSIIaJ ) significantly reduce SSIIa activity, resulting in an increase in amylopectin short chains with degree of polymerization (DP) ? 12 compared to indica rice cultivars (SSIIaI ). 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 EM204 (ss2a) from a screen of ca. 1,500 plants of the rice cultivar Kinmaze (japonica) that were subjected to N-methyl-N-nitrosourea mutagenesis. The SSIIa gene in EM204 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 EM204 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 EM204 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 EM204 rice grains was higher than that in its parent Kinmaze. EM204 also had higher content of amylopectin short chains (DP ? 12) than Kinmaze, which reduced the gelatinization temperature of EM204 starch by 5.6°C compared to Kinmaze. These results indicate that EM204 starch will be suitable for making foods and food additives that easily gelatinize and slowly retrograde.

Dataset Information

Analyses of starch biosynthetic protein complexes and starch properties from developing mutant rice seeds with minimal starch synthase activities.

Publications

Analyses of starch biosynthetic protein complexes and starch properties from developing mutant rice seeds with minimal starch synthase activities.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets