Characterization of pullulanase (PUL)-deficient mutants of rice (Oryza sativa L.) and the function of PUL on starch biosynthesis in the developing rice endosperm

Abstract

Rice (Oryza sativa) allelic sugary1 (sug1) mutants defective in isoamylase 1 (ISA1) accumulate varying levels of starch and phytoglycogen in their endosperm, and the activity of a pullulanase-type of a debranching enzyme (PUL) was found to correlate closely with the severity of the sug1 phenotype. Thus, three PUL-deficient mutants were generated to investigate the function of PUL in starch biosynthesis. The reduction of PUL activity had no pleiotropic effects on the other enzymes involved in starch biosynthesis. The short chains (DP < or = 13) of amylopectin in PUL mutants were increased compared with that of the wild type, but the extent of the changes was much smaller than that of sug1 mutants. The alpha-glucan composition [amylose, amylopectin, water-soluble polysaccharide (WSP)] and the structure of the starch components (amylose and amylopectin) of the PUL mutants were essentially the same, although the average chain length of the B(2-3) chains of amylopectin in the PUL mutant was approximately 3 residues longer than that of the wild type. The double mutants between the PUL-null and mild sug1 mutants still retained starch in the outer layer of endosperm tissue, while the amounts of WSP and short chains (DP < or = 7) of amylopectin were higher than those of the sug1 mutant; this indicates that the PUL function partially overlaps with that of ISA1 and its deficiency has a much smaller effect on the synthesis of amylopectin than ISA1 deficiency and the variation of the sug1 phenotype is not significantly dependent on the PUL activities.

Fig. 1.

Sites of Tos17 insertion and mutation site by MNU in the OsPUL gene and determination of the Tos17 rice mutant line genotype by PCR. (A) Structure of the OsPUL gene. The exons and introns are depicted as grey and white boxes, respectively. ATG and TGA indicate the translation initiation and stop codons, respectively. The sites of Tos17 insertion and mutation by MNU in mutant lines are indicated with a vertical arrow. Horizontal half arrows show the sites of primers (T1F, T2F, 5F, 6F, 10F, 11F, 9R, 10R, 11R, and 13R) for PCR for genotype determination (B) and mutant line screening. The primers T1F and T2F were designed from the Tos17 sequence, while 5F, 6F, 10F, 11F, 9R, 10R, 11R, and 13R were designed from the OsPUL gene sequence. The region used as probes for Southern blotting to screen mutant lines is indicated. (B) Determination of genotype [homozygous for Tos17 insertion (–/–, left panel) or wild homozygous (+/+, right panel)] in Tos17 mutant lines by nested PCR. Primer pairs are indicated below the photographs. ‘T1F/10F-T2F/11F’ means that the primer pair T1F/10F was used for the 1st PCR and T2F/11F for the 2nd PCR. M, molecular markers.

Fig. 2.

Native-PAGE/activity staining and immunoblotting of developing endosperm in rice PUL (pullulanase) mutant lines and the wild type. (A) Native-PAGE/debranching enzyme (DBE) activity staining (upper panel) and immunoblotting (lower panels) of rice developing endosperm of the wild type (T65) from day after flowering (DAF)-3 to -35. Two lanes of native-PAGE in each DAF were from the soluble fraction of the two independent developing rice endosperms. The ISA (isoamylase), PUL, and PHO (phosphorylase) bands are indicated by arrowheads. (B) Native-PAGE/PUL activity staining. PUL activity bands are indicated by arrowheads. (C) Immunoblotting using antiserum raised against purified rice PUL (Nakamura et al., 1996). (D) Native-PAGE/DBE activity staining. The ISA, PUL, and PHO activity bands are indicated by arrowheads. (E) Native-PAGE/branching enzyme (BE) activity staining. The BEI, BEIIa, and BEIIb activity bands are indicated by arrowheads. (F) Native-PAGE/starch synthase (SS) activity staining. The SSIIIa and SSI activity bands are indicated by arrowheads. Nip, Nipponbare; e10–/– and i16–/–,PUL mutant lines of Nip by Tos17 insertion; T65, Taichung 65; EM1003: PUL mutant of T65 induced by chemical mutagenesis. e10+/+ and i16+/+ are the control lines of e10–/– and i16–/–, respectively.

Fig. 3.

(A) Elution profiles by gel filtration chromatography through Sephacryl S-1000SF of Nipponbare (Nip, black line) and e10–/– (grey line). (B, C) Chain-length distributions of starch (B) and amylopectin (C) from Nip (left) and e10–/– (right). Solid line, fluorescence; short dashed line, refractive index; dashed and dotted line, DP; number with arrowhead, peak DP.

Fig. 4.

(A) Differences in the chain-length distribution patterns of endosperm amylopectin in the mature endosperm of PUL mutant lines (e10–/–, i16–/–, and EM1003), ISA mutant lines (EM914 and EM653), and the wild-type parent cultivars ‘Nip’ and ‘T65’. (B) Magnification of the pattern of PUL mutant lines in (A). Values for mole% of PUL mutant lines in (A) and (B) for each DP are averages of three (e10–/– and i16–/–) or two (EM1003) seeds arbitrarily chosen from a single homozygous plant. Relative SDs of the mole% of each chain length from DP6-30 was less than 8.3%.

Fig. 5.

Frequency dependence of the storage modulus (G’) and loss modulus (G'’) for the 4wt% endosperm starch paste in Nipponbare and e10–/– at 50 °C.

Fig. 6.

Characterization of the double mutant lines, #4001 (A, C, E) and AMF60 (B, D, F), in PUL mutant lines (e10–/– and EM1003) and ISA mutant lines (EM653 and EM914), respectively, and the wild type, Nipponbare (Nip), and Taichung65 (T65). (A, B) Native-PAGE/DBE activity staining. The ISA, PUL, and PHO activity bands are indicated by arrowheads. (C, D) Stereo micrographs of the cross-sections of mature endospern stained by iodine solution. (E, F) Differences in the chain-length distribution patterns of endosperm amylopectin in the nature endosperm between the double mutan lines #4001 (E) and AMF60 (F), the PUl mutant lines e10–/– (E) and EM914 (F), and the ISA mutant lines EM653 (E) and EM914 (F) and the wild-type parent cultivars.