Function and characterization of starch synthase I using mutants in rice

Abstract

Four starch synthase I (SSI)-deficient rice (Oryza sativa) mutant lines were generated using retrotransposon Tos17 insertion. The mutants exhibited different levels of SSI activities and produced significantly lower amounts of SSI protein ranging from 0% to 20% of the wild type. The mutant endosperm amylopectin showed a decrease in chains with degree of polymerization (DP) 8 to 12 and an increase in chains with DP 6 to 7 and DP 16 to 19. The degree of change in amylopectin chain-length distribution was positively correlated with the extent of decrease in SSI activity in the mutants. The structural changes in the amylopectin increased the gelatinization temperature of endosperm starch. Chain-length analysis of amylopectin in the SSI band excised from native-polyacrylamide gel electrophoresis/SS activity staining gel showed that SSI preferentially synthesized DP 7 to 11 chains by elongating DP 4 to 7 short chains of glycogen or amylopectin. These results show that SSI distinctly generates DP 8 to 12 chains from short DP 6 to 7 chains emerging from the branch point in the A or B(1) chain of amylopectin. SSI seemingly functions from the very early through the late stage of endosperm development. Yet, the complete absence of SSI, despite being a major SS isozyme in the developing endosperm, had no effect on the size and shape of seeds and starch granules and the crystallinity of endosperm starch, suggesting that other SS enzymes are probably capable of partly compensating SSI function. In summary, this study strongly suggested that amylopectin chains are synthesized by the coordinated actions of SSI, SSIIa, and SSIIIa isoforms.

Figure 1.

Characterization of SSI protein in rice developing endosperm. A, Separation of the SSI and SSIIIa activity peaks of rice developing endosperm using HiTrapQ anion-exchange chromatography. Each fraction was assayed for SS activity under two different conditions, either in a reaction buffer containing 0.5 m citrate and lacking glycogen ([C(+)G(−)], black circles and thin line) or in a reaction buffer lacking citrate and containing 2 mg/mL glycogen ([C(−)G(+)], white circles and bold line). B, Native-PAGE/SS activity staining of each fraction in A. The gel contains 0.1% rice amylopectin. The black and white arrowheads indicate putative SSI and SSIIIa bands, respectively. CE, Crude enzyme extract; FT, flow through fraction; number 1 to approximately 15, fraction numbers as in A. C, Native-PAGE/SS activity staining of rice developing endosperm from DAF 9 to 25. Fifteen percent of pooled SP (see “Materials and Methods”) was loaded on each lane. The gel contains 0.1% rice amylopectin. Black arrowheads indicate putative SSI and SSIIIa bands. These activity bands were dependent on the addition of ADP-Glc in the incubation buffer (data not shown). D, Amount of SSI or GBSSI protein in developing rice endosperm from DAF 12 to 30. Proteins of developing endosperm were divided into three fractions: SP, LBP, and TBP (see “Materials and Methods”). The amount of SSI or GBSSI protein was quantified by immunoblotting using antiserum raised against SSI or GBSSI. The data are the mean ± se of three seeds. The numbers on the graph are the relative SSI amount (%) of each protein fraction in the developing endosperms.

Figure 2.

Sites of Tos17 insertion in the OsSSI gene and determination of rice mutant lines genotype by PCR. A, Structure of the OsSSI gene. The exons and introns are depicted as black and gray boxes, respectively. ATG and TGA indicate the translation initiation and stop codons, respectively. The insertion site of Tos17 in the four mutant lines (names in boxes) are indicated by vertical arrows. Horizontal half arrows show the sites of primers for PCR for genotype determination (B) and mutant lines screening. The primers T1R, T2R, T1F, and T2F were designed from the Tos17 sequence while 1F, 2F, 3F, 6F, 1R, 2R, 4R, and 5R were designed from the OsSSI gene sequence. The region used as probe for Southern blotting to screen mutant lines is indicated. B, Determination of genotype (homozygous for Tos17 insertion [−/−, left section] or wild homozygous [+/+, right section]) in the four mutant lines by nested PCR. Primer pairs are indicated below the photographs. T1R, T2R/1F, 2F means that the primer pair T1R/1F was used for the first PCR, and T2R/2F for the second PCR. Nip, Rice cultivar Nipponbare (the wild-type parent of the mutant lines); M, molecular markers.

Figure 3.

Estimation of SSI activity and amount of SSI protein in rice SSI mutants. A, Native-PAGE/SS activity staining to estimate SSI activity in mutants (−/−) and their respective control lines (+/+). The numbers above the lanes are the volumes (microliters) of crude enzyme extract applied onto each lane. The SSIIIa and SSI activity bands are indicated by arrowheads. B, SDS-PAGE (left section, stained with Coomassie Brilliant Blue) and immunoblotting (right section) of SP, LBP, and TBP from developing endosperm using antiserum raised against rice SSI for quantification of the amount of SSI protein in Nipponbare and the SSI mutant lines. M, Molecular markers.

Figure 4.

Characterization of the SSI mutant line e7−/−, its control line e7+/+, and their wild-type parent cultivar Nipponbare. A, Seed morphology. B, X-ray diffraction pattern of endosperm starch. C, SEM of endosperm starch. Bar = 5 μm.

Figure 5.

A, Chain-length distribution patterns of endosperm amylopectin in mature endosperm of mutant lines (−/−) and the wild-type parent cultivar Nipponbare. B and C, Differences in chain-length distribution patterns of endosperm amylopectin between the mature endosperm of mutants (−/−) and their respective control lines (+/+; B), and in developing endosperm at DAF 7, 16, and 25 of mutant line e7−/− and Nipponbare (C). Values for molar % in A and Δ molar % in B and C for each DP are averages of three seeds arbitrarily chosen from a single homozygous plant. Vertical bars in B and C indicate ses. The numbers on the plots are DP values. Inset in C shows differences in chain-length distribution of amylopectin at different endosperm developmental stages in Nipponbare.

Figure 6.

Physicochemical properties of starch granules in mature endosperm of rice SSI mutant lines. A, Differences in peak temperature of thermal gelatinization (°C) as measured by DSC between each mutant line (−/−) and its respective control (+/+). The y axis gives the temperature (°C) difference ([−/−] − [+/+]). The values presented are average ± se of at least three seeds arbitrarily chosen from a single homozygous plant. B, Pasting properties of endosperm starch of the mutant line e7−/− and its control line e7+/+. The viscosity value at each temperature point is the average of three replications. The thin line indicates the change in temperature during measurement by rapid visco analyzer.

Figure 7.

Chain-length distribution of polyglucans from SS bands excised from the native-PAGE/SS activity staining gel of the wild-type rice cultivar Nipponbare. Polyglucans were extracted from SSI and SSIIIa activity bands and from a portion without any activity bands. The SS activity band gel segment contained modified substrate whereas the control gel fragment contained unmodified substrate only. A, Chain-length differences between the unmodified substrate (oyster glycogen gel, Oysgel) and the SSI-modified (SSI-band) or SSIIIa-modified (SSIIIa-band) substrate. The SP fraction from developing endosperm was used for native-PAGE/SS activity staining. B, Chain-length differences between the unmodified rice amylopectin (rice amylopectin gel, Rice AP gel) and the SSI-modified rice amylopectin substrate (SSI-band). The partially purified SSI fraction (Fig. 1B, lane 4) was used for native-PAGE/SS activity staining on a gel containing rice amylopectin. Chains with DP 4 to 5 are considered as products of digestion of polyglucans by hydrolytic enzyme(s) included in the SSI fraction during prolonged incubation time (20 h). The superimposed black line shows the difference in amylopectin chain-length distribution between the mature endosperm SSI mutant line e7−/− and its control line e7+/+.

Figure 8.

A, Schematic representation of the proposed model for the elongation of rice amylopectin glucan chains by SSI and other SS isozymes. Circles represent Glc residues. In the wild type, A and B₁ chains grow through the addition of two to six Glc residues (black circles) by SSI. Black and gray circles in A and B₁ chains are elongated by other SS isozymes when SSI is deficient. The double circle marks the point in the B₁ chain where a branch (A chain) emerges. The A chains and the exterior parts of B chains (from nonreduced end to branch point), both ranging from DP 12 to DP 16 in length (Hizukuri, 1986), compose the crystalline domain of amylopectin clusters. The length of one cluster of amylopectin corresponds to DP 27 to 28 (Hizukuri, 1986). In waxy maize, Bertoft (1991, 2004) estimated the total internal chain length of amylopectin to be DP 12.4. If this value holds true for rice amylopectin, the length of the B₁ chain would be the combined length of the exterior part plus DP 11. The partially broken arrows labeled “elongation by other SS isozymes” indicate compensatory function of other SS isozymes when SSI is deficient. B, The chain-length distribution (molar %) of endosperm amylopectin of control line (e7+/+; section a) and the difference in chain-length distribution (Δ molar %) of endosperm amylopectin between the SSI mutant (e7−/−) and the control line (e7+/+; section b). These data are from Figure 5B. C, The rate of molar changes of each chain relative to the amount of its chain (Δ molar %/molar % × 100) as calculated from Figure 8B sections a and b, for DP 5 to 60 amylopectin chains of SSI mutant (e7−/−). The changes in DP 6 to 7 and DP ≥ 23 chain regions where molar percentages are low (Fig. 8B, a) are emphasized. The zones for the A, B₁, and B₂ chain were estimated along with the x axis from the result of rice amylopectin described in Hizukuri (1986). Insets are the schematic representation of the chain structure in the amylopectin molecule. D, Schematic representation of the proposed range of glucan chains acted upon (solid line) and synthesized (broken line) by SSI, SSIIa, and SSIIIa in developing rice endosperm based on our present results on SSI, previous data on SSIIa (Nakamura et al., 2005), and our present results and those of Jane et al. (1999) on SSIIIa, respectively. Regions of overlaps define the range of glucan chains that could possibly be synthesized by SSIIa and/or SSIIIa in compensation for SSI deficiency.