Starch biosynthesis in rice endosperm requires the presence of either starch synthase I or IIIa

Abstract

Starch synthase (SS) I and IIIa are the first and second largest components of total soluble SS activity, respectively, in developing japonica rice (Oryza sativa L.) endosperm. To elucidate the distinct and overlapping functions of these enzymes, double mutants were created by crossing the ss1 null mutant with the ss3a null mutant. In the F(2) generation, two opaque seed types were found to have either the ss1ss1/SS3ass3a or the SS1ss1/ss3ass3a genotype. Phenotypic analyses revealed lower SS activity in the endosperm of these lines than in those of the parent mutant lines since these seeds had different copies of SSI and SSIIIa genes in a heterozygous state. The endosperm of the two types of opaque seeds contained the unique starch with modified fine structure, round-shaped starch granules, high amylose content, and specific physicochemical properties. The seed weight was ∼90% of that of the wild type. The amount of granule-bound starch synthase I (GBSSI) and the activity of ADP-glucose pyrophosphorylase (AGPase) were higher than in the wild type and parent mutant lines. The double-recessive homozygous mutant prepared from both ss1 and ss3a null mutants was considered sterile, while the mutant produced by the leaky ss1 mutant×ss3a null mutant cross was fertile. This present study strongly suggests that at least SSI or SSIIIa is required for starch biosynthesis in rice endosperm.

Fig. 1.

Pedigree of opaque seeds of the TO and WO lines and seed morphology. The morphology of rice dehulled seeds was observed using a stereo-microscope with overhead light (upper panels) and on a light box (lower panel). (This figure is available in colour at JXB online.)

Fig. 2.

Determination of genotypes in opaque seeds of the TO and WO lines by nested PCR. The left figures show the site of Tos17 insertion and the position of the primer pairs. Horizontal half arrows show the binding sites for nested PCR primers used for genotype determination (T1FT2F, 1R2R, and 3F6F for the SSI gene, and T1RT2R, 5F6F, and 5R6R for the SSIIIa gene). Primer pairs are indicated below the photographs. ‘T1FT2F/1R2R’ means that the primer pair T1F/1R was used for the first PCR and T2F/2R for the second PCR. M, molecular markers. (This figure is available in colour at JXB online.)

Fig. 3.

Native-PAGE/SS activity staining of self-pollinated developing endosperm of ss1ss1/SS3ass3a in the TO line (A) and SS1ss1/ss3ass3a in the WO line (B) and their chain length distribution. Fourteen developing endosperm samples were randomly chosen, and the zymogram pattern of native-PAGE/SS activity staining was compared with those of Nipponbare, the ss1 mutant (ΔSSI, e7), and the ss3a mutant (ΔSSIIIa, e1). SSIIIa (A) or SSI (B) activity bands in 14 developing endosperm samples were divided into two groups, namely higher or lower activity bands, respectively, compared with Nipponbare. Lane numbers with circles indicate endosperm having the same or a slightly lower SSIIIa (A) or SSI (B) activity band, respectively, than that of Nipponbare. Chain length distributions of endosperm starch of a typical lower or higher activity band (boxed lanes) were analysed (bold lines) and compared with those of ΔSSI (thin line) and ss1ss1/SS3ass3a (dotted line) (A), or ΔSSIIIa (thin line) and SS1ss1/ss3ass3a (dotted line) (B). (This figure is available in colour at JXB online.)

Fig. 4.

Amylopectin structure of ss1ss1/SS3ass3a and SS1ss1/ss3ass3a. (A) Chain length distribution patterns of endosperm amylopectin in the mature endosperm of ss1ss1/SS3ass3a, the ss1 mutant (e7, ΔSSI), and the wild-type parent cultivar Nipponbare. (B) Chain length distribution patterns of endosperm amylopectin in the mature endosperm of SS1ss1/ss3ass3a, the ss3a mutant (e1, ΔSSIIIa), and the wild-type parent cultivar Nipponbare. (C) Differences in the chain length distribution patterns of amylopectin in the mature endosperm of ss1ss1/SS3ass3a and SS1ss1/ss3ass3a and their parent mutant lines. The inset in C shows the magnification of the pattern in the range of chains with DP 30–60. The numbers on the plots are the DP values.

Fig. 5.

Size separation of debranched endosperm starch and purified amylopectin from ss1ss1/SS3ass3a and SS1ss1/ss3ass3a, ss1 mutant (ΔSSI, e7), ss3a mutant (ΔSSIIIa, e1), and wild-type Nipponbare by gel filtration chromatography through Toyopearl HW55S–HW50S columns. Elution profiles of isoamylase-debranched starch (black lines) and purified amylopectin (grey lines) are shown. Each fraction (Fr. I, II, and III) was divided into the valley of the carbohydrate content curve determined by the phenol sulphuric acid method.

Fig. 6.

Characterization of the endosperm starch in the ss1ss1/SS3ass3a, SS1ss1/ss3ass3a, ss1 mutant (ΔSSI, e7), ss3a mutant (ΔSSIIIa, e1), and wild-type Nipponbare. (A) X-ray diffraction patterns of endosperm starch. The height and sharpness of major peaks show the degree of starch granule crystallinity. (B) Pasting properties of endosperm starch by rapid visco-analyser (RVA). The viscosity pattern shown is one of at least three replicates. The thin line indicates the change in temperature during measurement with an RVA. (C) Scanning electron micrographs of the endosperm starch granules. Bar=5 μm. (This figure is available in colour at JXB online.)

Fig. 7.

Amount of SSI or GBSSI protein in the mature endosperm of ss1ss1/SS3ass3a, SS1ss1/ss3ass3a, ss1 mutant (ΔSSI, e7), ss3a mutant (ΔSSIIIa, e1), and wild-type Nipponbare. The total protein amount in the three fractions [SP (soluble protein)+LBP (loosely bound protein) and TBP (tightly bound protein) of SSI or GBSSI protein] was quantified by immunoblotting using antiserum raised against SSI or GBSSI (Fujita et al., 2006). The data are the mean ±SE of three seeds. Asterisks denote statistically significant differences between Nipponbare and mutant lines by t-test at P <0.05.

Fig. 8.

Pedigree, seed morphology (A), native-PAGE/SS activity staining of developing endosperm (B), and differences in the amylopectin chain length distribution pattern (C) of ss1^Lss1^L/ss3ass3a after a cross between the leaky ss1 mutant (ΔSSI, i2-1) and the ss3a mutant (ΔSSIIIa, e1). The morphology of rice dehulled seeds was observed using a stereo-microscope with overhead light (left panel) and on a light box (right panels). Lane numbers with circles on the zymogram (B) show WO endosperm having the same level or a slightly lower SSI activity band than that of Nipponbare.