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, 121 (2), 399-410

The Starch-Debranching Enzymes Isoamylase and Pullulanase Are Both Involved in Amylopectin Biosynthesis in Rice Endosperm

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The Starch-Debranching Enzymes Isoamylase and Pullulanase Are Both Involved in Amylopectin Biosynthesis in Rice Endosperm

A Kubo et al. Plant Physiol.

Abstract

The activities of the two types of starch debranching enzymes, isoamylase and pullulanase, were greatly reduced in endosperms of allelic sugary-1 mutants of rice (Oryza sativa), with the decrease more pronounced for isoamylase than for pullulanase. However, the decrease in isoamylase activity was not related to the magnitude of the sugary phenotype (the proportion of the phytoglycogen region of the endosperm), as observed with pullulanase. In the moderately mutated line EM-5, the pullulanase activity was markedly lower in the phytoglycogen region than in the starch region, and isoamylase activity was extremely low or completely lost in the whole endosperm tissue. These results suggest that both debranching enzymes are involved in amylopectin biosynthesis in rice endosperm. We presume that isoamylase plays a predominant role in amylopectin synthesis, but pullulanase is also essential or can compensate for the role of isoamylase in the construction of the amylopectin multiple-cluster structure. It is highly possible that isoamylase was modified in some sugary-1 mutants such as EM-273 and EM-5, since it was present in significant and trace amounts, respectively, in these mutants but was apparently inactive. The results show that the Sugary-1 gene encodes the isoamylase gene of the rice genome.

Figures

Figure 1
Figure 1
A, Light micrographs of cross-sections through kernels at the late-milking stage of rice sugary-1 mutants EM-5 and EM-41 and their parent cv Kinmaze. Sections were stained with iodine solution. B, Schematic representation for partitioning the phytoglycogen (PG) region and the starch region of sugary endosperm.
Figure 2
Figure 2
Size separation of polyglucans in various endosperm regions from the sugary-1 mutants EM-5 and EM-914 by Sephacryl S-1000 gel-filtration chromatography. After chromatography, a part of each fraction was used for the measurement of carbohydrate content by the phenolic sulfuric method (○), and the other part (0.8 mL) of the fraction was added to 0.2 mL of 0.1% I2/1% KI solution for the measurement of the λmax (fine line). A, B, C, and D show results for samples prepared from sections a, b, c, and d, respectively, which were partitioned from the EM-5 endosperm as illustrated in Figure 1B. E and F show results from the whole endosperms of cv Kinmaze and EM-914, respectively.
Figure 3
Figure 3
Comparison of chain length distribution of polyglucans in the phytoglycogen and starch regions of mature endosperm of sugary-1 mutants and their parent cultivars. Polyglucans were debranched by P. amyloderamosa isoamylase and reduced with sodium borohydrate. The distribution of the reduced linear glucan chains was determined by HPAEC-PAD analysis. A, Polyglucans from the starch regions of EM-5, EM-41, and EM-935 were prepared from the Sephacryl S-1000 chromatogram (fraction nos. 12–15) as shown in Figure 2. B, Polyglucans from the phytoglycogen regions of mutants EM-5, EM-273, EM-914, and EM-935 prepared as shown in Figure 1B (as d). Normal polyglucans (amylopectin) in endosperm of wild-type cv Kinmaze and cv Taichung-65 were prepared from the whole kernels without embryo (shown in A and B). C, Comparison of the results between polyglucans prepared from the starch region of EM-5 endosperm partitioned as shown in Figure 1B (as a) and those from Sephacryl S-1000 chromatogram (fraction nos. 12–15) as shown in Figure 2A. D, Difference in the distribution of chain lengths of polyglucans between wild-type amylopectin from cv Kinmaze and polyglucans from the starch region of EM-5 (black bar; data from Fig. 3A) and between wild-type amylopectin and polyglucans from the phytoglycogen region of EM-5 (white bar; data from Fig. 3B).
Figure 4
Figure 4
Distribution of pullulanase activity in cross-section of endosperm at the late-milking stage from sugary-1 mutants and their parent cultivars as detected by the pullulan-film method. Incubation time was 30 min and Red Pullulan content was 3%.
Figure 5
Figure 5
Activities of isoamylase in the endosperm at the late-milking stage of various sugary-1 mutants and their parent cultivars. Isoamylase activity visualized as blue bands were detected by native-PAGE/activity staining method. A, Results with partially purified isoamylase preparations as described in “Materials and Methods.” Lane 1, cv Kinmaze; lane 2, EM-5; lane 3, EM-41; lane 4, EM-273; lane 5, cv Taichung-65; lane 6, EM-914; lane 7, EM-935. Samples (5 μL) applied onto the 7.5% (w/v) polyacrylamide gel. B, Results from partially purified isoamylase samples prepared as described in “Materials and Methods” from endosperms of EM-5 and EM-41 and their parent cv Kinmaze. Volumes of samples applied onto the gel: 15 μL for lanes 1, 5, and 8; 3 μL for lanes 2, 6, and 9; 1.5 μL for lanes 3, 7, and 10; 0.75 μL for lane 4. Lanes 1 through 4, cv Kinmaze; lanes 5 through 7, EM-5; lanes 8 through 10, EM-41. C, Results from crude extracts of the inner and the outer regions of EM-5 and EM-41 endosperms fractionated as described in “Materials and Methods.” Lanes 1 through 3, Inner region of EM-5 endosperm; lanes 4 through 6, outer region of EM-5 endosperm; lanes 7 through 9, inner region of EM-41 endosperm; lanes 10 through 12, outer region of EM-41 endosperm. Volumes of samples applied onto the 7.5% polyacrylamide gel: 15 μL for lanes 1, 4, 7, and 10; 5 μL for lanes 2, 5, 8, and 11; 1 μL for lanes 3, 6, 9, and 12. Arrows indicate the bands corresponding to isoamylase (ISA), pullulanase (PUL), and amylases (AMY), respectively.
Figure 6
Figure 6
Western-blot analysis of isoamylase in endosperm of various sugary-1 mutants and their parent cvs at the late-milking stage. The isoamylase preparation was partially purified as in Figure 5A, and 10 μL each of the sample was applied on the SDS-polyacrylamide (10%) gel, followed by immunoblot detection with polyclonal antibodies raised against purified isoamylase from developing rice endosperm (Fujita et al., 1999). Lane 1, cv Kinmaze; lane 2, EM-5; lane 3, EM-41; lane 4, EM-273; lane 5, cv Taichung-65; lane 6, EM-914; lane 7, EM-935.

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