Starch-branching enzyme I-deficient mutation specifically affects the structure and properties of starch in rice endosperm

Abstract

We have isolated a starch mutant that was deficient in starch-branching enzyme I (BEI) from the endosperm mutant stocks of rice (Oryza sativa) induced by the treatment of fertilized egg cells with N-methyl-N-nitrosourea. The deficiency of BEI in this mutant was controlled by a single recessive gene, tentatively designated as starch-branching enzyme mutant 1 (sbe1). The mutant endosperm exhibited the normal phenotype and contained the same amount of starch as the wild type. However, the mutation apparently altered the fine structure of amylopectin. The mutant amylopectin was characterized by significant decrease in both long chains with degree of polymerization (DP) > or = 37 and short chains with DP 12 to 21, marked increase in short chains with DP < or = 10 (A chains), and slight increase in intermediate chains with DP 24 to 34, suggesting that BEI specifically synthesizes B1 and B2-3 chains. The endosperm starch from the sbe1 mutant had a lower onset concentration for urea gelatinization and a lower onset temperature for thermo-gelatinization compared with the wild type, indicating that the genetic modification of amylopectin fine structure is responsible for changes in physicochemical properties of sbe1 starch.

Figure 1.

Effects of sbe1 (BEI-deficient) mutation on the expression of BEI in endosperm. A, SDS-PAGE profile of the crude protein extract of rice endosperm in mature rice kernels. B, Native-PAGE/activity staining of BEs in developing seed of rice. The migration and identification of each band corresponding to three BE isoforms (BEI, BEIIa, and BEIIb) and phosphorylase were according to our previous report (Yamanouchi and Nakamura, 1992). The volumes of crude enzyme extracts applied were 0.67 μL. C, Western-blot analysis of BEI in mature rice kernels. The immunoblot was developed with antiserum raised against BEI from rice endosperm (Nakamura et al., 1992) at a dilution of 1/1000. D, Northern-blot analysis of BEI transcripts in rice endosperm. Total RNA from developing grains was blotted and probed with the specific RNA probe, EST clone EST#17(EC0727).

Figure 2.

Kernel of rice sbe1 mutant. Left to right: rice cv T65 (wild type); EM557S (sbe1 mutant); and EM529 (ae mutant).

Figure 3.

Gene dosage effects of sbe1 mutation on BE isoforms of rice endosperm. A, SDS-PAGE profile of the crude protein extract of rice endosperm in mature rice kernels. B, Western-blot analysis of BEI and BEIIb in rice endosperm. Protein was extracted from 20 mg of mature rice powder. The immunoblot was developed with antiserum raised against BEI or BEIIb from rice endosperm (Nakamura et al., 1992) at a dilution of 1/1000.

Figure 4.

Effect of sbe1 mutation on chain length distribution of amylopectin in endosperm from BEI-deficient mutant of rice as determined by APTS-capillary electrophoresis. Chain length distributions were determined by analyzing APTS-labeled debranched starches on the molar basis, and the peak area of a fraction of linear chain with a specific chain length was calculated as a percentage of total peak area up to DP of 80, although the figures present the results up to DP 60 only. A, The distribution of α-1,4-glucan chains in amylopectin from wild-type rice cv T65 and the four homozygous sbe1 mutant lines, BMF69, BMF70, BMF71 (EM557S), and EMF22. The data for rice cv T65 were the averages of those for three different homozygous rice cv T65 lines, although no substantial differences among them were detected. B, The chain distribution of amylopectin from two sbe1/wx lines (EMF25 and EMF26) and two wx mutant lines (BMF23-1 and BMF23-2). C, The chain distribution of amylopectin from rice cv T65 and ae mutant EM529. The data for rice cv T65 were the averages of those for three different homozygous rice cv T65 lines. Figures on the right show differences between the respective mutants and rice cv T65 (A and C) or the wx mutant line BMF23-1 (B). The data shown are representatives from three experiments that gave similar results. For isolation of homozygous mutant and wild-type lines, see “Materials and Methods” in detail.

Figure 5.

Effect of sbe1 mutation on gelatinization properties of starch from endosperm of mature seed. A, Gelatinization of starch from the sbe1 mutant and wild type in various concentrations of urea solution. Ten milligrams of rice powder in an Eppendorf tube was mixed with 1 mL of urea solution and shaken for 24 h at 25°C. After centrifugation, samples were allowed to stand for 1 h. B, Absorbance spectra of resolved starches in the I₂/KI solution. These starches were obtained in the supernatant after treatment with 4 m urea solution as shown by asterisks in A.