Hikaru Satoh

Mutation of the Plastidial α-Glucan Phosphorylase Gene in Rice Affects the Synthesis and Structure of Starch in the Endosperm

Plastidial phosphorylase (Pho1) accounts for ∼96% of the total phosphorylase activity in developing rice (Oryza sativa) seeds. From mutant stocks induced by N-methyl-N-nitrosourea treatment, we identified plants with mutations in the Pho1 gene that are deficient in Pho1. Strikingly, the size of mature seeds and the starch content in these mutants showed considerable variation, ranging from shrunken to pseudonormal. The loss of Pho1 caused smaller starch granules to accumulate and modified the amylopectin structure. Variation in the morphological and biochemical phenotype of individual seeds was common to all 15 pho1-independent homozygous mutant lines studied, indicating that this phenotype was caused solely by the genetic defect. The phenotype of the pho1 mutation was temperature dependent. While the mutant plants grown at 30°C produced mainly plump seeds at maturity, most of the seeds from plants grown at 20°C were shrunken, with a significant proportion showing severe reduction in starch accumulation. These results strongly suggest that Pho1 plays a crucial role in starch biosynthesis in rice endosperm at low temperatures and that one or more other factors can complement the function of Pho1 at high temperatures.

MNU-induced mutant pools and high performance TILLING enable finding of any gene mutation in rice

Mutant populations are indispensable genetic resources for functional genomics in all organisms. However, suitable rice mutant populations, induced either by chemicals or irradiation still have been rarely developed to date. To produce mutant pools and to launch a search system for rice gene mutations, we developed mutant populations of Oryza sativajaponica cv. Taichung 65, by treating single zygotic cells with N-methyl-N-nitrosourea (MNU). Mutagenesis in single zygotes can create mutations at a high frequency and rarely forms chimeric plants. A modified TILLING system using non-labeled primers and fast capillary gel electrophoresis was applied for high-throughput detection of single nucleotide substitution mutations. The mutation rate of an M2 mutant population was calculated as 7.4xa0×xa010−6 per nucleotide representing one mutation in every 135xa0kb genome sequence. One can expect 7.4 single nucleotide substitution mutations in every 1xa0kb of gene region when using 1,000xa0M2 mutant lines. The mutations were very evenly distributed over the regions examined. These results indicate that our rice mutant population generated by MNU-mutagenesis could be a promising resource for identifying mutations in any gene of rice. The modified TILLING method also proved very efficient and convenient in screening the mutant population.

A Novel Factor FLOURY ENDOSPERM2 Is Involved in Regulation of Rice Grain Size and Starch Quality

The authors clone the rice FLOURY ENDOSPERM2 (FLO2) gene; flo2 mutants have aberrant endosperm, and FLO2 overexpressors have enlarged grains. Gene expression and protein interaction studies indicate that FLO2, a novel tetratricopeptide repeat containing protein, regulates storage starch and protein gene expression in rice endosperm development and may also play a role in heat tolerance. Rice (Oryza sativa) endosperm accumulates a massive amount of storage starch and storage proteins during seed development. However, little is known about the regulatory system involved in the production of storage substances. The rice flo2 mutation resulted in reduced grain size and starch quality. Map-based cloning identified FLOURY ENDOSPERM2 (FLO2), a member of a novel gene family conserved in plants, as the gene responsible for the rice flo2 mutation. FLO2 harbors a tetratricopeptide repeat motif, considered to mediate a protein–protein interactions. FLO2 was abundantly expressed in developing seeds coincident with production of storage starch and protein, as well as in leaves, while abundant expression of its homologs was observed only in leaves. The flo2 mutation decreased expression of genes involved in production of storage starch and storage proteins in the endosperm. Differences between cultivars in their responsiveness of FLO2 expression during high-temperature stress indicated that FLO2 may be involved in heat tolerance during seed development. Overexpression of FLO2 enlarged the size of grains significantly. These results suggest that FLO2 plays a pivotal regulatory role in rice grain size and starch quality by affecting storage substance accumulation in the endosperm.

Rice endosperm-specific plastidial α-glucan phosphorylase is important for synthesis of short-chain malto-oligosaccharides

Previous genetic studies have indicated that the type L alpha-glucan phosphorylase (Pho1) has an essential role during the initiation process of starch biosynthesis during rice seed development. To gain insight into its role in starch metabolism, we characterized the enzymatic properties of the Pho1 recombinant form. Pho1 has significantly higher catalytic efficiency toward both linear and branched alpha-glucans in the synthesis direction than in the degradation direction with equilibrium constants for the various substrates ranging from 13 to 45. Pho1 activity is strongly inhibited by its own reaction product (Pi) in the synthesis reaction (K(i)=0.69 mM) when amylopectin is the primer substrate, but this inhibition is less pronounced (K(i)=14.2 mM) when short alpha-glucan chains are used as primers. Interestingly, even in the presence of Pi alone, Pho1 not only degrades maltohexaose but also extends them to synthesize longer MOSs. Production of a broad spectrum of MOSs (G4-G19) was stimulated by both Pi and Glc1P in an additive fashion. Thus, even under physiological conditions of high Pi/Glc1P, Pho1 extends the chain length of short MOSs which can then be used as subsequent primer by starch synthase activities. As ADP-glucose strongly inhibits Pho1 activity, Pho1 likely operates only during the initial stage and not during maturation phase of starch synthesis.

Structure, physical, and digestive properties of starch from wx ae double-mutant rice.

Amylopectin is the principal component of starch. The amylose extender (ae) gene encodes the starch-branching enzyme IIb, which is critical in determining the fine structure of endosperm starch. To determine the relationship between the fine structure of amylopectin and its physical properties, rice mutant lines defective in the ae function with altered fine structure of amylopectin and in combination with the waxy (wx) background were selected for comparative studies with primary wild-type and ae starches. The ae mutant endosperms accumulated a high amylose content starch with long amylopectin chains. The ae and wx ae starches showed no significant difference in the unit chain-length distribution of amylopectin and starch granule morphology. The wx ae starch displayed a higher pasting temperature and higher peak viscosity. The gelatinization peak temperatures of the wx, ae, and wx ae starches were 2.2, 13.1, and 17.1 degrees C higher, respectively, than that of the wild-type starch, and the wx ae starch showed a retrogradation peak with a shorter cooling period than that of ae starch. The raw ae and wx ae starches were almost indigestible by alpha-amylase in vitro. Rats fed the wx ae starch showed slowly increasing blood glucose at a lower level than the rats fed the wx or wild-type starch. These results indicate that the primary structure of the rice wx ae amylopectin with enriched long chains changes the granular structure of the starch, including its crystal structure, and results in resistance to in vitro or in vivo degradation.

Protein Body Biogenesis in Cereal Endosperms

Developing cereal endosperms accumulate storage proteins as a reserve to support the early postgerminating phase of plant growth and development. These storage proteins are concentrated and packaged into a specialized organelle, the protein body, which are derived from the endoplasmic reticulum lumen or vacuole. In this chapter, we describe the biochemical and cellular events that underlies the basis for the use of these two intracellular sites for storage protein deposition and accumulation.

Development of Coenzyme Q10-Enriched Rice Using Sugary and Shrunken Mutants

Coenzyme Q10 (CoQ10) is a popular food supplement. Earlier, we successfully produced CoQ10 in rice, which normally produces predominately CoQ9. Here we developed efficient production of CoQ10 in rice by introducing the gene for decaprenyl diphosphate synthase into rice sugary and shrunken mutants. These rices produced 1.3 to 1.6 times as much CoQ10 as the earlier enriched rice did.

Genetic variation of glutelin acidic subunit polypeptides in Bangladesh rice genetic resources

Bangladesh rice genetic resources collected from six distinct regions of the country were examined to obtain the diversity in glutelin acidic subunit polypeptides. Seed glutelins from 576 Bangladesh rice cultivars representing seven ecotypes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF) electrophoresis and two-dimensional electrophoresis (2-DE: SDS-PAGE/IEF) analyses. Glutelin acidic subunit was separated into four bands, α-1, α-2, α-3 and α-4, and the variation in each of bands was detected by SDS-PAGE analysis. A higher molecular size component of α-1(α-1′) was identified in the cultivars tested. In case of α-2, α-2H with high molecular mass, α-2L with low molecular mass and α-2H/L were detected. α-3 band variation showed α-3H, α-3L, and α-3H/L, while for α-4, α-4H, α-4L bands were identified. In IEF analysis, a total of 16 bands with independent pI ranging from pI 6.30 to 7.52 were identified for the glutelin acidic subunit among the cultivars. The maximum and minimum numbers of IEF bands found were 13 and 9, respectively. The α-2L less cultivars were also lacking in pI 6.80 polypeptide in IEF. Result of 2-DE showed that pI 6.80 polypeptide was the main component of α-2L band. Transplanted Aman ecotype was the most diverse with respect to glutelin variation. Geographical distribution of glutelin variation in the Transplanted Aman ecotype differed according to the regions. The above results indicate that Bangladesh rice cultivars possess great genetic diversity in glutelin acidic subunit polypeptides. This study of indigenous rice cultivars from Bangladesh provides useful information regarding their breeding potential.