Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 137 (1), 43-56

Complementation of sugary-1 Phenotype in Rice Endosperm With the Wheat isoamylase1 Gene Supports a Direct Role for isoamylase1 in Amylopectin Biosynthesis

Affiliations

Complementation of sugary-1 Phenotype in Rice Endosperm With the Wheat isoamylase1 Gene Supports a Direct Role for isoamylase1 in Amylopectin Biosynthesis

Akiko Kubo et al. Plant Physiol.

Abstract

To examine the role of isoamylase1 (ISA1) in amylopectin biosynthesis in plants, a genomic DNA fragment from Aegilops tauschii was introduced into the ISA1-deficient rice (Oryza sativa) sugary-1 mutant line EM914, in which endosperm starch is completely replaced by phytoglycogen. A. tauschii is the D genome donor of wheat (Triticum aestivum), and the introduced fragment effectively included the gene for ISA1 for wheat (TaISA1) that was encoded on the D genome. In TaISA1-expressing rice endosperm, phytoglycogen synthesis was substantially replaced by starch synthesis, leaving only residual levels of phytoglycogen. The levels of residual phytoglycogen present were inversely proportional to the expression level of the TaISA1 protein, although the level of pullulanase that had been reduced in EM914 was restored to the same level as that in the wild type. Small but significant differences were found in the amylopectin chain-length distribution, gelatinization temperatures, and A-type x-ray diffraction patterns of the starches from lines expressing TaISA1 when compared with wild-type rice starch, although in the first two parameters, the effect was proportional to the expression level of TaISA. The impact of expression levels of ISA1 on starch structure and properties provides support for the view that ISA1 is directly involved in the synthesis of amylopectin.

Figures

Figure 1.
Figure 1.
Complementation of sugary-1 phenotype of EM914 in rice endosperm transformed by introduction of the TaISA1 gene. A, Representation of the BAC construct insert containing the TaISA1 gene. The introduced TaISA1 gene was detected by PCR method as described in “Materials and Methods.” NPT, Neomycin phosphotransferase; LB, left border; RB, right border; M, DNA size markers; N, nontreated control line; P, pCLD04541. B, Fluorescence in situ hybridization of a probe for the wheat DNA including the TaISA1 gene (red as indicated by arrows) and the 1S marker probe (green) with the transgenic rice (line 47-3) chromosomes. C, Screening strategy. The complementation of sugary-1 phenotype was shown by the accumulation of iodine-stained starch granules in endosperm from the TaISA1 transformant seeds at the nonsegragated T1 generation since phytoglycogen in the host sugary-1 mutant line, EM914, did not react with iodine while wild-type cv Kinmaze contained iodine-stained starch granules. D, Staining of cross sections of rice kernel with iodine solution. Mature sugary-1 (EM914) seeds were shrunken and did not stain with iodine solution due to a complete replacement of starch by phytoglycogen, whereas wild-type seeds contained dark-brown starch granules. Note that the homozygous seed phenotypes in some transformed lines (6-1, 24-2, and 47-3) at the T2 generation containing starch-like polyglucans were similar to the wild type.
Figure 2.
Figure 2.
Expression and activities of ISA isoforms. A, Expression of OsISA1, OsISA2, and OsISA3 in developing endosperm and young green leaves of wild-type rice cv TC65 as measured by the RT-PCR method. B, Expression of OsISA1 and OsISA2 in developing endosperm at the early milking stage of the TaISA1 transgenic lines, TC65, and EM914 as measured by the RT-PCR method. C, Western-blot analysis of OsISA1 in developing endosperm at the late-milking stage of the TaISA1 transgenic lines, TC65, EM914, and wild-type wheat using polyclonal antibodies raised against purified OsISA1 (Fujita et al., 1999). Note that the anti-OsISA1 did not react to TaISA1. D, Expression of TaISA1 in developing endosperm at the early milking stage of the TaISA1 transgenic lines, TC65, EM914, and wild-type wheat as measured by the RT-PCR method. E, Western-blot analysis of ISA1 in developing endosperm at the late-milking stage of the TaISA1 transgenic lines, TC65, and wild-type wheat using polyclonal antibodies raised against purified TaISA1. Note that the anti-TaISA1 reacted not only to TaISA1 but also to OsISA1, and that the molecular size of the cross-reacted OsISA1 band was slightly larger than those of TaISA1 bands in wheat and the TaISA1 transgenic lines of rice. The data are the results of three separate measurements using separate single seeds. F, Native-PAGE activity staining of starch debranching enzymes in developing endosperm at the late-milking stage of transgenic lines (11-2, 26-1, 24-2, and 47-3), TC65, and EM914. The soluble protein extract was separated on a polyacrylamide gel containing 0.3% (w/v) amylopectin. After electrophoresis, the gel was incubated at 30°C for 2 h and stained with iodine solution. The positions of the ISA and pullulanase activity bands stained blue.
Figure 3.
Figure 3.
Detection of enzymes involved in starch biosynthesis in developing endosperm at the early milking stage of the TaISA1 transgenic lines, TC65, and EM914. A, Native-PAGE staining of BE isoforms BEI, BEIIa, and BEIIb. Soluble proteins were separated in native-PAGE gel. The gel was incubated in a solution containing phosphorylase a for 5 h at 30°C and then added by an iodine solution. These three BE activity bands stained purple. B, Native-PAGE staining of SS isoforms. Soluble proteins were separated in native-PAGE gels containing 0.8% (w/v) of oyster glycogen, incubated in the reaction solution containing 0. 5 m sodium citrate, and added by an iodine solution. The SSI and SSIIa activity bands stained dark blue. C, Western-blot analysis of pullulanase (PUL), BEI, and BEIIb with polyclonal antibodies raised against the respective enzymes purified from developing rice endosperm.
Figure 4.
Figure 4.
Chain-length distribution of amylopectin and total polyglucans in mature endosperm of the TaISA1 transgenic lines, TC65, and EM914. A, Normalized chain-length distribution of total polyglucans as percentage for each peak area of the total areas of all peaks with 3 ≤ DP ≤ 60. B, Differential plot derived by subtracting molar percentages of wild-type TC65 polyglucans from those of transformants and sugary-1 mutant EM914 (shown in A). C, Differential plot similar to B, except that amylopectin separated from soluble polyglucans by centrifugation was used instead of total polyglucans, as described by Fujita et al. (2003). Note that the data are similar between B and C, although amounts of very short chains with DP ≤ 8 in total polyglucans of line 24-2 (B) were higher to some extent than those in purified amylopectin (C), possibly due to the WSP included in total polyglucan preparation.
Figure 5.
Figure 5.
X-ray diffraction pattern of starch in endosperm of the TaISA1 transgenic lines and TC65.
Figure 6.
Figure 6.
Scanning electron micrographs of starch granules in endosperm of the TaISA1 transgenic lines and TC65. Starches were purified as described in “Materials and Methods.” Labels are TC65 (A and B), 47-3 (C and D), 6-1 (E and F), and 24-2 (G and H). The bars in A to H = 5 μm.
Figure 7.
Figure 7.
Comparison of chromatographic behavior of ISA between rice and wheat. Electrophoretic mobility of ISA activities on native gel containing 0.3% amylopectin was detected by staining of polyglucans with iodine solution. A, The ISA preparations were partially purified from 20 developing endosperm of TC65 and the TaISA1 transformant line 47-3 and 40 endosperm of wild-type wheat (W). The ISAI activity bands are indicated by arrows. B, The Hitrap-Q column chromatography of the ISA activity from developing endosperm of TC65, line 47-3, and wheat. The number on top of each lane indicates fraction number. c, The crude enzyme extract from TC65 endosperm. C, The Hitrap-Q column chromatography of the ISA activity from a mixture of developing endosperm of TC65 and wheat with the same volume. The number on top of each lane means fraction number.
Figure 8.
Figure 8.
Relationship between levels of DBE and the structure of amylopectin, the WSP content, and onset gelatinization temperature (T0). A, The ISA protein content and the activity of pullulanase. The values are means ± sd of nine separate measurements using nine separate single seeds, respectively. The data include the results shown in Figure 2E. B, The percentage of short chains with DP ≤ 12 of polyglucans. The data include the results shown in Table III. C, The content of WSP. The data are obtained from the results shown in Table II. D, The T0 values. The data are obtained from the results shown in Table III.

Similar articles

See all similar articles

Cited by 27 articles

See all "Cited by" articles

LinkOut - more resources

Feedback