Starch consists mainly of amylose and amylopectin. It occurs as water insoluble particles in the form of “starch granules” in plant tissue, and is generally classified into three types (A, B, and C) according to the wide-angle X-ray diffraction (WAXD) patter n given by their amylopectin cr ystalline structures. Cereal starch, including rice starch, had an A-type WAXD pattern, tuber starch a B-type, and bean and root starch a C-type (a mixture of A- and B-types) [1]. A genetic analysis using amylopectin mutants showed that a specific gene was impor tant in determining the amylopectin structure and crystal pattern. The amylose-extender ( ae) mutant, with a defect in the starch branching enzyme IIb function, accumulated starch which had a reduced ability to gelatinize due to a change in the fine structure of amylopectin with enriched long chains. The powder WAXD analysis of purified ae starch showed a B-type diffraction pattern, whereas wild-type rice starch showed an A-type. The sugary1 mutant, with reduced isoamylase 1 activity, accumulated water-soluble polyglucans named as phytoglycogen. The phytoglycogen contained more short-glucan chains than wild type amylopectin, in contrast to the enriched longer glucan chains in ae amylopectin. In some sugary1 lines, endosperm cells were clearly separated into a starch region and a phytoglycogen region as evidenced by iodine staining [2]. The localization of starch granules in the kernel is important, because it influences the gelatinization properties of rice. Powder WAXD analysis, which was typically used to characterize the crystalline structure of starch, tells us nothing about localization because the starch for this analysis is generally extracted and purified from tissue, destroying localization information and possibly damaging the starch granules. The aim of this study was to visualize starch localization and the starch crystalline structure by microbeam WAXD analysis. We used mutant lines generated by treating of fer tilized egg cells of japonica rice cv Kinmaze (wild type) with N-methyl- N-nitrosourea [3]. The use of double mutants that include a waxy ( wx) defective gene ensures that the starch consists of essentially amylopectin, which makes up most of its crystal structure. The mature kernels from ae, wx double mutant ( wx / ae) and sugary1, wx double mutant ( wx / sugar y1) were used as materials. The experiments were performed at beamline BL40XU with a high-flux beam ( λ = 0.083 nm) of 5 μ m in diameter. WAXD patterns were recorded using an image intensifier coupled to a cooled CCD camera [4]. 0.2 mm and 0.02 mm thick slices were cut from rice kernels using a cryostat, and the crystalline structure was scanned sequentially in constant intervals as shown by the arrows in Fig. 1. Microbeam WAXD analysis visualized the starch localization and cr ystalline structure (Fig. 2). Amylose-free starches in the native rice kernels of the wx and wx/ae mutants showed A- and B-type WAXD patterns, respectively, with no difference between the outer and inner regions of the patterns. In contrast, the wx/sugary1 mutant kernel showed an A-type diffraction pattern in the outer region and amorphous in the inner region. The higher intensity at small angles in the outer regions of wx/sugary1 (scanning positions near 0 and near 1600) is likely due to scattering derived from density fluctuations in sugary amylopectin clusters. A chain-length distribution analysis of polyglucans in wild type and mutant kernels showed that ae amylopectin had more long chains and fewer short chains than the wild type and waxy amylopectin. In the wx/sugary1 kernel, the phytoglycogen in the inner region had many more short chains than the amylopectin in the outer region had. These results indicate that the branch chain length in polyglucans is crucial in determining the starch crystalline structure [5]. Several lines of ae mutants with mutations at same locus but with varying phenotypes have been isolated previously. The starch from ae mutant line EM16 was a typical B-type detected by powder WAXD but that from EM129 was C-type. When microbeam WAXD analysis was applied to the EM129 kernel, 48 T Th he e U Us se e o of f M Mi ic cr ro ob be ea am m X X- -R Ra ay y D Di if ff fr ra ac ct ti io on n f fo or r t th he e C Ch ha ar ra ac ct te er ri iz za at ti io on n o of f C Cr ry ys st ta al l S St tr ru uc ct tu ur re e o of f R Ri ic ce e S St ta ar rc ch h Life Science : Medical Biology 0 0 1 2 3 4 5 6 2 3 1 Position X (mm) Position Y (mm) Fig. 1. A rice kernel slice of wild type under the microscope. WAXD profiles were scanned linearly (a) or two-dimensionally (b) . Akiko Kubo a , Yoshiaki Yuguchi b and Shinichi Kitamura a, * a Graduate School of Life and Environmental Sciences, Osaka Prefecture University b Faculty of Engineering, Osaka Electro-Communication University *E-mail: skita@bioinfo.osakafu-u.ac.jp 49 References [1] Hizukuri et al .: Nature 192 (1961) 239. [2] Kubo et al .: Plant Physiol. 121 (1999) 399. [3] Satoh et al .: J. Fac. Agr. Kyushu Univ. 24 (1979)165. [4] Ohta et al .: J. Appl. Cryst. 38 (2005) 274. [5] A. Kubo, Y. Yuguchi, M. Takemasa, S. Suzuki, H. Satoh, S. Kitamura: J. Cereal Sci. 48 (2008) 92. EM16, position 2325 (B type) WT, position 150 (A type) EM129, position 0143 (Cb type) EM129, position 0016 (Ca type) Fig. 3. Microbeam WAXD patterns detected using thin slices (20 μ m). Diffraction patterns were recorded as shown in (b) of Fig. 1. White and red arrows show B- and A-type specific signals, respectively. C-type patterns from an ae kernel of EM129 were classified as Ca- and Cb-type. Fig. 2. Microbeam WAXD profiles of rice kernel starch granules. Diffraction patterns were recorded sequentially at intervals of 25 μ m along the arrow (a) in Fig. 1. Scanning Position ( μ m) Scanning Position ( μ m) Scanning Position ( μ m) wx wx/sugary1 wx/ae 2460 2700 2250 1800 1350 900 450 400 3000 1800 1500 1200 900 600 300 2280 1824 1368 912 456 2500 2000 1500 1000 500 0 0 800 1200 1600 1968 1476 984 492 2 4 6 8 10 12 14 16 18 20 22 2 4 6 8 10 12 14 16 18 20 22 2 4 6 8 10 12 14 16 18 20 22 0 0 0 0 Diffraction Intensity Diffraction Intensity Diffraction Intensity 2 (deg.) θ 2 (deg.) θ 2 (deg.) θ C-type diffraction patterns scanned at different positions were classified to Ca- and Cb-type (Fig. 3). This result agreed with the idea that the C-type is the mixture of A- and B-type crystalline structures [1]. We did not observe any pattern in the regional distribution of Ca- and Cb-type starch. In conclusion, microbeam WAXD is a useful method to map the distribution of starch crystalline structures in the rice kernel.
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