The structure of starch can be manipulated by changing. the expression levels of starch branching enzyme IIb in rice endosperm

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1 Plant Biotechnology Journal (2004) 2, pp doi: /j x The structure of starch can be manipulated by changing Blackwell Publishing, Ltd. the expression levels of starch branching enzyme IIb in rice endosperm Naoki Tanaka 1, Naoko Fujita 2,3, Aiko Nishi 4, Hikaru Satoh 4, Yuko Hosaka 3, Masashi Ugaki 5, Shinji Kawasaki 1 and Yasunori Nakamura 2,3, * 1 National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Ibaraki , Japan 2 Department of Biological Production, Akita Prefectural University, Shimoshinjo-Nakano, Akita-City , Japan 3 Japan Science Technology, Kawaguchi, Saitama , Japan 4 Department of Agriculture, Kyushu University, Hakozaki, Higashi-ku, Fukuoka , Japan 5 Department of Agriculture, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, Japan Received 12 April 2004; revised 11 May 2004; accepted 14 May *Correspondence (fax ; nakayn@akita-pu.ac.jp) Keywords: amylopectin, endosperm, starch, starch branching enzyme, transgenic rice. Summary When the starch branching enzyme IIb (BEIIb) gene was introduced into a BEIIb-defective mutant, the resulting transgenic rice plants showed a wide range of BEIIb activity and the fine structure of their amylopectins showed considerable variation despite having the two other BE isoforms, BEI and BEIIa, in their endosperm at the same levels as in the wild-type. The properties of the starch granules, such as their gelatinization behaviour, morphology and X-ray diffraction pattern, also changed dramatically depending on the level of BEIIb activity, even when this was either slightly lower or higher than that of the wild-type. The over-expression of BEIIb resulted in the accumulation of excessive branched, water-soluble polysaccharides instead of amylopectin. These results imply that the manipulation of BEIIb activity is an effective strategy for the generation of novel starches for use in foodstuffs and industrial applications. Introduction Starch branching enzymes (BEs) play an essential role in starch biosynthesis by introducing α-1,6-glucosidic linkages into α-1,4-glucosidic chains. Branching by BEs is specific as the branches in amylopectin are regularly arranged and account for amylopectin tandem-cluster structure ( Thompson, 2000; Nakamura, 2002; Blennow et al., 2004). This structure is responsible for the differences in physicochemical properties between starch and glycogen, and variations in the structure are thought to be responsible for the differences between the starches of a variety of plant species, tissues and genetic backgrounds. Plants have two types of BE, historically designated as BEI and BEII in cereals (Boyer and Preiss, 1978) or as type B and type A in legumes and tubers, respectively (Burton et al., 1995). Attempts have been made to alter the amylopectin fine structure of potato tubers by manipulating BEs through antisense technology (Safford et al., 1998) or by introducing bacterial BEs (Kortstee et al., 1996). Changes in the expression of the genes encoding BEI and BEII were found to affect the physicochemical properties of tuber starches (Safford et al., 1998; Jobling et al., 1999; Schwall et al., 2000). Dicotyledonous plant tissues, such as pea embryo (Burton et al., 1995), kidney bean embryo (Hamada et al., 2001) and potato tuber (Larsson et al., 1996), have only a single BEIItype isoform, which has also been referred to as A-type BE in the literature. On the other hand, two BEII isoforms, BEIIa and BEIIb, have been identified in the endosperm of cereals such as maize (Boyer and Preiss, 1978), rice (Mizuno et al., 1992; Nakamura et al., 1992), barley (Sun et al., 1997) and wheat (Morell et al., 1997). BEIIb is specifically expressed in the endosperm of maize (Fisher et al., 1996; Gao et al., 1997), rice ( Yamanouchi and Nakamura, 1992; Mizuno et al., 1993) and barley (Sun et al., 1998), whereas BEIIa is present in all organs examined (Yamanouchi and Nakamura, 1992). It is 2004 Blackwell Publishing Ltd 507

2 508 Naoki Tanaka et al. likely that BEIIb plays an essential role in determining cereal-specific starch granule amylopectins, as these exhibit the A-type X-ray diffraction pattern, whereas tuber starches show the B-type pattern (Hizukuri, 1996; Jane et al., 1997). BEIIb-deficient rice mutants, referred to as amylose-extender (ae) mutants, produce shrivelled seeds due to the accumulation of abnormal intermediate-sized starches (Baba and Arai, 1984), with reduced ability to gelatinize (Nishi et al., 2001). Nishi et al. (2001) showed that the alteration of starch properties in rice endosperm is caused by the change in the fine structure of amylopectin with enriched long chains within a cluster. These studies indicate that BEIIb influences the fine structure of amylopectin by producing short chains, and that BEIIa, as well as BEI, cannot perform this function ( Nakamura, 2002; Satoh et al., 2003a,b). We report here, for the first time, a method for the production of novel starches by the introduction of the BEIIb gene in cereal endosperm depleted in native BEIIb gene. The highlight of the present study is that the manipulation of gene expression can produce a wide range of different starches with a variety of distinct structural, physicochemical and morphological properties. The functional roles of the BEIIb gene in the manufacture of the novel starches in the endosperm of transgenic rice plants and the possible applications are also discussed. Results Generation of transgenic rice plants Transgenic rice plants with various expression levels of the BEIIb protein were produced by introducing a normal BEIIb gene from the Japonica-type rice cultivar, Shimokita, into the ae mutant EM10, which was derived from Japonica rice cv. Kinmaze (Figure 1A). We selected six homozygous lines Figure 1 (A) OsBEIIb gene structure, and construct used for transformation. The gene was cloned into pcambia. The clone contained the promoter region up to 2239 bp upstream from the transcription starting site and the transcribed region for the BEIIb gene, where the position of the initiation codon (ATG) was at +127 bp from the transcription starting site. White boxes indicate exons. Cleavage sites of common restriction enzymes (B, BamHI; E, EcoRI; H, HindIII; P, Pst I; S, SalI) are indicated above the map. The probe for Southern analysis (B) is shown below. RB, right T-DNA border; LacZ, LacZ alpha gene; P35S, CaMV35S promoter; HTPII, hygromycin resistance gene; CaMV35S polya, CaMV35S polya terminator; LB, left T-DNA border. (B) Southern blot analysis of genomic DNA from transformants and their parent cultivars. DNA preparations (3 µg each) were digested with HindIII and electrophoresed on 0.8% agarose gel. The bands of approximately 10 kb (*) and 6 kb (**) are evidently the endogenous and introduced BEIIb fragments, respectively. (C) Northern blot analysis of BEIIb, BEI, BEIIa, SSI (starch synthase I), SSIIIa, ISA1 (isoamylase1) and PUL (pullulanase) in transformants and their parent cultivars. The lower portion of the figure shows the ethidium bromide-stained RNA gel. (D) Western blot analysis of BEI and BEIIb in developing rice kernels at the late-milking stage of transformants and their parent cultivars. Branching enzyme (BE) isoforms are indicated by arrowheads. (E) Native polyacrylamide gel electrophoresis (PAGE)/activity staining of BE isoforms in immature rice kernels of transformants and their parent cultivars. The data shown in (B E) are representative experimental results.

3 Manipulation of starch structure 509 (#1-1, #7-8, #9-8, #31-1, #106-1 and #113-7) from 50 individual T 0 progeny lines, whose seeds in the T 2 generation had amylopectin chain length profiles in their endosperm that differed from those of the ae mutant EM10, as described in Experimental procedures. Phenotypic analyses were conducted on seeds from the homozygous lines in the T 3 or T 4 generation. The replications shown in the tables and figures of the present study were performed by multiple experiments using different kernels arbitrarily chosen from a pool of kernels harvested from a single homozygous T 3 or T 4 plant. So far examined, the specific phenotypes in each homozygous line were consistent amongst separate plants and were maintained unchanged in the succeeding generations. Southern and Northern blot analyses demonstrated that the introduced BEIIb gene was expressed in the transformants, with the exception of line #7-8, which was used as a control (Figure 1B,C). Western blot analysis showed that the amount of BEIIb protein varied between the transgenic lines (Figure 1D), and corresponded to the BEIIb activity as measured by native polyacrylamide gel electrophoresis (PAGE) staining (Figure 1E). The copy number of the introduced BEIIb genes seemingly increased with increases in the BEIIb protein expressed (Figure 1B,D). The genetic analysis suggests that the BEIIb gene was tandem introduced into transformed line #1-1 (data not shown). The BEIIb protein was only expressed in endosperm in transgenic lines as in the wild-type (data not shown). These results indicate that the approximately 2.2 kb regulatory region of the BEIIb gene used in this study contains the full promoter activity of the gene. There was no significant change in the activities of BEI and BEIIa or in the levels of their transcripts (Figure 1C E), and no pleiotropic effects on starch synthase I and IIIa, isoamylase 1 or pullulanase (Figure 1C), in all transformants tested. Figure 2 Phenotypes of transgenic rice seeds and properties of their starch. (A) Whole seeds (upper panel) and their cross-sections (lower panel). (B) Gelatinization properties of starch as indicated by the solubility in urea solution. (C) Structure of starch granules as revealed by scanning electron microscopy. Bar, 5 µm. (D) X-Ray diffraction pattern of starch granules of transformants and their parent cultivars. Phenotypic changes in the rice kernels of the ae transformants The ae mutation in EM10 results in kernels of decreased size and chalky appearance (Figure 2A; Nishi et al., 2001). When the BEIIb activity in the transformed line was very low and comparable with that of EM10 (see transformant #7-8), the dry kernels resembled those of EM10 (Figures 1A and 2A). With an increase in BEIIb activity, the appearance and size of the kernels (#9-8, #106-1, #31-1 and #113-7) approached those of Kinmaze (Figures 1E and 2A; Table 1); when the BEIIb activity was greatly in excess of that in Kinmaze, the kernel became strikingly wrinkled (#1-1; Figures 1E and 2A; Table 1). When the total polyglucans were centrifuged at 600 g for 20 min, the proportion of soluble polyglucans was markedly Table 1 Weight of hulled grain, polyglucan content per grain and level of soluble polyglucan in the endosperm of transgenic plants and their parents Line Dehulled grain* (mg) Total polyglucan (mg) Soluble polyglucan (%) Kinmaze 23.7 ± ± ± 0.1 EM1O 17.9 ± ± ± 0.1 # ± ± ± 0.8 # ± ± ± 0.8 # ± ± ± 0.6 # ± ± ± 0.4 # ± ± ± 0.8 # ± ± ± 0.2 *Values are the means ± standard error of 20 seeds. Values are the means ± standard error of at least three replications.

4 510 Naoki Tanaka et al. higher in #1-1 (about 10.8%) than in the other transformants (3.7% 5.4%) ( Table 1). The results suggest that water-soluble polysaccharides (WSPs) were produced in #1-1 endosperm, as found in the isoamylase 1-deficient sugary-1 mutant of rice (Wong et al., 2003). The structure of the endosperm amylopectin and the physicochemical properties of the starch in ae transformants To examine the effect of the BEIIb level on the fine structure of the endosperm amylopectin, the chain length distribution of debranched polyglucans on a molar basis in the transformants was measured by high-resolution fluorescence-labelled capillary electrophoresis. As the BEIIb activity increased, short chains of degree of polymerization (DP) 13 became more frequent, whilst the numbers of long chains of DP = and DP 40 declined (Figure 3). It is striking that this specific effect was maintained when BEIIb levels were higher than those in Kinmaze. The sizes of soluble and insoluble polyglucans in the endosperm of transformant #1-1 were smaller than those of the peak endosperm fraction (Fr. 19) of phytoglycogen in the sugary-1 mutant EM935, as well as those of the amylopectin fraction (Fr. 12) of Kinmaze (Figure 4A,B). The chain profiles of the debranched polyglucans of both the soluble and insoluble peak fractions of #1-1 suggested that they had a distinctive structure with more highly branched polyglucans than in amylopectin, but less than in phytoglycogen (Figure 4C). Overall, the results indicate that BEIIb plays a role in forming short chains of amylopectin clusters, and that its activity level profoundly influences the fine structure of amylopectin, even when its activity exceeds that in the wild-type, despite the fact that the other BE isoforms, BEI and BEIIa, are still present at the same levels as in the wild-type (Figure 1C E). The change in the structure of amylopectin dramatically affected the gelatinization and other physicochemical properties of the starch granules as well as their morphology. Figure 2B shows that the swelling of the starch granules in 4 M urea increased with BEIIb activity, and that the polyglucans of #1-1 were almost completely dissolved. Scanning electron micrographs showed that the ae starch granules were much more variable in size than the polygonal granules of the wildtype (Figure 2C), and, as the BEIIb activity of the transformants increased, the shape and size of their starch granules approached those of the wild-type; however, in #1-1, most of the starch granules were decomposed into small particles. Table 2 shows that the BEIIb activity dramatically affected the thermal properties of the starch as measured by differential scanning calorimetry (DSC). Thus, the temperatures of onset of gelatinization (T o ) were 69.2, 66.1, 61.2, 55.4, 52.6 and 42.8 C for transformants #7.8, #9.8, #106.1, #31.1, #113.7 and #1.1, respectively, whereas those of the ae and Kinmaze starches were 69.6 and 56.5 C, respectively. Table 2 Thermal properties of the starch granules in transgenic rice endosperm Line T o * ( C) T p ( C) T c ( C) H (mj/mg) EM ± ± ± ± 0.26 # ± ± ± ± 0.56 # ± ± ± ± 0.23 # ± ± ± ± 1.04 Kinmaze 56.5 ± ± ± ± 0.90 # ± ± ± ± 0.79 # ± ± ± ± 0.09 # ± ± ± ± 0.22 Figure 3 Chain length distribution of polyglucans in rice endosperm. (A) Comparison of the chain length distribution of total polyglucans in transformants and their parent cultivars. (B) Differences in chain length distribution of total polyglucans in transformants and EM10 relative to Kinmaze. *Onset temperature. Peak temperature. Conclusion temperature. Gelatinization enthalpy of starch. Values are the means ± standard error of at least three replications.

5 Manipulation of starch structure 511 Figure 4 Size and fine structure of soluble and insoluble polyglucans from rice endosperm. (A) Size separation of soluble polyglucans (#1-1S, open triangle) and insoluble polyglucans (#1-1P, filled circle) from #1-1 by Sephacryl S-1000SF gel filtration chromatography. (B) Size separation of soluble polyglucans of EM935 (sugary-1 mutant, EM935S, open diamond) and insoluble polyglucans of Kinmaze (KinP, filled square). The fractions (Fr.) whose chain profiles are analysed in (C) are indicated. (C) Comparison of the chain length distributions of polyglucans from Fr. 22 of line #1-1S, Fr. 22 of #1-1P, Fr. 19 of EM935S and Fr. 12 of KinP, obtained by gel filtration chromatography in (A) and (B), respectively. It is known that rice endosperm starch gives the A-type X- ray diffraction pattern, in contrast with the B-type diffraction pattern of the ae starch, as shown in Figure 2D. Interestingly, the starches of transformants #106-1, #31-1 and #113-7 exhibited a typical A-type pattern, whereas that of #7-8 displayed a typical B-type pattern and #9-8 an intermediate pattern. Moreover, the #1-1 starch formed no clear-cut crystal structure. These results indicate that the level of BEIIb activity dramatically influences the crystalline structure of starch. Discussion The application of antisense technology to BE genes has been used to produce novel starches with modified amylopectin in potato tubers (Safford et al., 1998; Jobling et al., 1999; Schwall et al., 2000; Jobling, 2004), although potato has only single isoforms of BEI type (B-type BE) and BEII type (A-type BE). Although the BEII type is only slightly expressed and the BEI type accounts for the bulk BE activity in potato tubers, the BEII type has a major effect on the structure of amylopectin (Jobling et al., 1999), whilst almost complete inhibition of BEI-type activity can only slightly modify amylopectin branch patterns in transgenic potato tubers (Safford et al., 1998). It is interesting to note that simultaneous inhibition of both types of BE has novel effects on tuber starch, in that the contents of amylose and phosphorus are dramatically elevated (Schwall et al., 2000). The strategy used in these studies can offer novel starches applicable for food and industrial uses. At the same time, however, these starches with altered amylopectin structure were only obtained using antisense technology in potato tubers. Starch structure and properties are wholly specific for plant species, and the composition and relative activities of BE isoforms markedly differ amongst plant species and tissues. The present study succeeded in modifying the structure and properties of starch in cereal endosperm by manipulating BE activities. It has been shown here that the manipulation of BEIIb gene expression of rice can generate starches exhibiting

6 512 Naoki Tanaka et al. Figure 5 Relationship between BEIIb protein level and the phenotypes of transformants and their parental lines. (A) The amount of BEIIb in a single developing endosperm at the late-milking stage was measured by Western blot analysis using polyclonal antibodies for purified BEIIb from rice endosperm (Nakamura et al., 1992). The mean values and SE were calculated from five independent experiments using arbitrarily chosen kernels from BEIIb transformants, ae mutant EM10 and wild-type cv. Kinmaze. (B) Mean values and SE of the proportion of short chains with DP 13 of polyglucans in rice endosperm, calculated from three independent experiments using arbitrarily chosen kernels from BEIIb transformants, EM10 and Kinmaze. (C) Mean values and SE of the onset gelatinization temperature of starch granules in rice endosperm, calculated from three independent experiments using arbitrarily chosen kernels from BEIIb transformants, EM10 and Kinmaze. The data are the same as those shown in Table 2. great variations in the fine structure of amylopectin ( Figure 3). It should be noted that the effect of BEIIb activity on the structure of amylopectin was entirely dependent on the level of BEIIb activity, and changes in amylopectin structure occurred at all activity levels, not only when the BEIIb activity was much lower or higher than that in the wild-type, but also when it was close to the wild-type level (Figure 1D,E). When BEIIb was expressed in the ae mutant, the proportion of short amylopectin chains of DP 13 increased and the proportion of long chains of DP = and DP 40 decreased (Figure 3). It should be noted that these changes reflect the levels of BEIIb activity, as the levels of BEI and BEIIa were essentially unchanged (Figure 1E). This finding indicates that BEIIb plays a unique role in the formation of A chains in the crystalline region of the amylopectin cluster (Nishi et al., 2001; Nakamura, 2002; Nakamura et al., 2003; Satoh et al., 2003a,b). BEIIb expression in the ae mutant also affected gelatinization ( Table 2 and Figure 2B), the X-ray diffraction pattern (Figure 2D) and morphology (Figure 2C) of the starch granules. Thus, the manipulation of BEIIb activity is useful for producing starches with different properties in rice endosperm. Figure 5 summarizes that the extent of BEIIb activity was positively correlated with the proportion of the short chains with DP 13 of amylopectin, which, in turn, was negatively correlated with the onset temperature for gelatinization (T o ) of starch. The alteration of BEIIb activity over a narrow range around the wild-type level affected the amylopectin structure (compare the changes in #106-1, #31-1 and #113-7 with the wild-type). It is remarkable that an excess of BEIIb activity

7 Manipulation of starch structure 513 hampered the cluster structure of amylopectin, as found in line #1-1 (Figures 1C E, 3A and 4). The polyglucan properties of #1-1 resembled those of phytoglycogen or WSP found in sugary-1 mutants (Sumner and Somers, 1944; Wong et al., 2003), but they were clearly different structurally from each other. In fact, line #1-1 starch exhibited distinct properties when compared with those of sugary-1 polyglucans. Firstly, most of the #1-1 polyglucans were not recovered in the soluble fraction (10.8%), in contrast with the phytoglycogen of the sugary-1 mutant (47.9%) (Fujita et al., 2003) and WSP of the antisense isoamylase transformants (16.2%) (Fujita et al., 2003). Secondly, both the chain profile and the size of the #1-1 polyglucan clearly differed from those of phytoglycogen (Figure 4). Thirdly, line #1-1 starch formed a granular structure to some extent (Figure 2C) and hence showed a lower T o value (Table 2) and a distorted X-ray diffraction pattern (Figure 2D), whereas sugary polyglucans did not show such a granular structure and did not have any measurable parameters for T o or X-ray diffraction pattern (Wong et al., 2003). It should be stressed that the amylopectin cluster structure in higher plant tissues can be decreased only under two conditions examined so far, i.e. under excessive activity of BEIIb, as observed in this study, or under isoamylase 1 deficiency, as reported in sugary-type mutants (see review by Satoh et al., 2003a). In this sense, the manipulation of BEIIb activity is an efficient way to change starch quality so that the functional properties of starch can be drastically altered. In the present study, the rice BEIIb gene was introduced into an ae mutant, EM10, defective in BEIIb protein. As a result, a series of transgenic lines produced a variety of starches in accordance with different expression levels of the BEIIb gene. This approach is useful, as the BEIIb gene is specifically expressed in the endosperm of rice and therefore the introduction of the gene has no physiological effects on the vegetative tissues of the plant. BEI is the predominant BE isoform, accounting for about 60 70% of the total BE activity, whilst the other two isoforms, BEIIa and BEIIb, share almost equally the remaining activity in rice endosperm, when assayed by the phosphorylase a stimulation assay (Yamanouchi and Nakamura, 1992). Our previous reports with three BE mutants strongly suggest the hierarchy of roles in amylopectin biosynthesis in rice endosperm amongst the three BE isoforms (Nishi et al., 2001; Nakamura, 2002; Satoh et al., 2003a, b): BEIIb plays a distinct role in forming short A chains within the cluster (Nishi et al., 2001), whereas BEI has a major effect on the synthesis of B 1 chain branches in the amorphous region of the cluster, as well as on the synthesis of cluster-linking B 2 B 4 chains (Satoh et al., 2003b). As no significant changes in amylopectin chain length profile are observed in the BEIIa-defective endosperm of rice, the function of the BEIIa isoform is considered to complement the above roles of BEIIb and BEI (Nakamura, 2002; Satoh et al., 2003a). These results suggest that there are at least two spatially distinct branches in the amylopectin cluster. One of the possible explanations is that the first group of branches is located in the amorphous region and the second group of branches is localized in the crystalline region, and that the latter branches are specifically synthesized by BEIIb, whereas the former branches are formed predominantly by BEI, although BEIIa and BEIIb can complement the function of BEI at least to some extent. If this hypothesis is true, BEIIb plays a major part in determining the number of total chains of a cluster by forming A chains in the crystalline region (Nakamura, 2002). This idea is consistent with the following experimental results: (i) the rice ae amylopectin has enriched B 2 B 4 chains as well as B 1 chains and depleted short A chains (Nishi et al., 2001); (ii) branches of amylopectin molecules from BEIIb-lacking potato tuber are almost localized in the amorphous region of the cluster, whereas those from wildtype cereal endosperm are located in both crystalline and amorphous regions, when examined by analysis of Naegeli dextrins from both starches ( Jane et al., 1997). As illustrated in Figure 5, the BEIIb activity level conferred a remarkable variation in starch gelatinization properties. When the BEIIb level was very low, starch was hardly gelatinized, with up to about 13 C higher T o values when compared with wild-type starch. These starches may be good materials for the paper industry because they can be easily removed by water. In contrast, when BEIIb was markedly expressed in excess of the wild-type level, the starch had a very low T o value. In line #1-1, a marked change in amylopectin structure transferred the starch to WSP-type polyglucans, with a marked decrease in the dry weight of the kernel (Table 1 and Figure 2A), although the fresh weight of the kernel was comparable with that of the wild-type during development (data not shown). This means that line #1-1 starch has a rather hydrophilic nature when compared with wild-type starch. This hydrophilic nature of #1-1 starch might be useful for preserving the water content or softness of food and industrial materials, as WSP has been added to some cosmetics for such a purpose. In conclusion, the present investigation indicates that the engineering of the BEIIb gene is a promising method for the development of starch granules with novel properties. For example, the structure and properties of polyglucans produced in line #1-1 cannot be replaced by naturally occurring starches. Such starches could be invaluable in foodstuffs and industrial materials.

8 514 Naoki Tanaka et al. Experimental procedures Plant material The BEIIb-deficient ae mutant line, EM10, of rice (Oryza sativa L.) was used (Nishi et al., 2001). It was derived from Japonica rice cv. Kinmaze by treatment with N-methyl-N-nitrosourea (Satoh and Omura, 1979). Cloning and sequencing of the BEIIb gene An 18-kb DNA fragment containing the promoter region and the transcribed region for the BEIIb gene was obtained from a rice bacterial artificial chromosome (BAC) library from green leaf protoplasts of cv Shimokita (Nakamura et al., 1997), and cloned into the SalI site of the binary vector pcambia1300 (a kind gift of Richard A. Jefferson of CAMBIA, Australia), as shown in Figure 1A. The fragment contained the 5 flanking region 2239 bp upstream of the BEIIb transcription start site. The resulting plasmid was designated pcbeiib. Preparation of total RNA from rice seeds and Northern blot analysis Preparation of total RNA from rice seeds and Northern blot analyses were performed according to Nishi et al. (2001). Extraction of BEs and staining of native PAGE gels for activity and Western blot analysis These procedures were carried out according to Nishi et al. (2001) and Kubo et al. (1999). The area of each band was digitized using an NIH image (ver. 1.55) program and then converted into the relative value. Measurement of gelatinization behaviour The gelatinization behaviour of mature seeds was examined in urea solution using the method developed by Nishi et al. (2001). DSC of starches was performed as described in Fujita et al. (2003). Transformation of the ae mutant and screening of transgenic lines Procedures for rice tissue culture and transformation with Agrobacterium tumefaciens were as described by Toki (1997). pcbeiib was transferred into A. tumefaciens EHA105 (Hood et al., 1993), and the transformants were introduced into EM10 calli. A total of 50 independent T 0 progeny lines were grown in a glasshouse maintained at 28 C by day (12 h) and 24 C by night (12 h) under natural daylight conditions. Ten randomly chosen T 1 seeds of 13 T 0 lines were grown and their T 2 seeds were examined for starch gelatinization properties in 4 M urea solution (Nishi et al., 2001), and BEIIb activities. Six lines with different gelatinization behaviours were selected and the respective homozygous lines (#1-1, #7-8, #9-8, #31-1, #106-1 and #113-7) were used for further study as transgenic lines. Lines were considered to be homozygous if all their T 2 seeds and the subsequent generation seeds obtained from the corresponding self-pollinated T 2 plant had the same pattern of chain length distribution of amylopectin. Preparation of genomic DNA from rice leaves and Southern blot analysis Genomic DNA extraction from rice leaves and Southern blot analysis were performed as described by Fujita et al. (2003). Determination of the distribution of α-polysaccharides by the capillary electrophoresis method Capillary electrophoresis of debranched starch was performed by a modification of the method of O Shea and Morell (1996), as described in Fujita et al. (2001), using the P/ACE MDQ Carbohydrate System (Beckman Coulter, CA, USA). Separation of soluble and insoluble α-polyglucans from rice endosperm At least three seeds from each transformant were examined to estimate soluble and insoluble polyglucans. The seeds were de-hulled with pliers, and powdered with a mortar and pestle. The powder was then washed three times with 1 ml of 85% methanol. The dried precipitate was resuspended in 0.5 ml of distilled water and stirred gently for 20 min at room temperature. The suspension was centrifuged at 600 g for 20 min at 20 C and the precipitate was dissolved in 0.5 ml of distilled water, stirred again for 20 min at room temperature and centrifuged. The pellet was washed with 0.5 ml of water and re-centrifuged. The supernatants were combined and centrifuged. The last supernatants, defined as soluble polyglucans, and the precipitates, defined as insoluble polyglucans, were used to estimate the amount of each type of polyglucan by the method of Fujita et al. (2003). About 6 g of seeds was used for the size separation of the α-polyglucan of #1-1, Kinmaze, EM10 and the sugary-1

9 Manipulation of starch structure 515 mutant (EM935; Wong et al., 2003) by Sephacryl S-1000SF chromatography and for scanning electron microscopy (SEM) observation and X-ray diffraction analysis of insoluble polyglucans of the transformant lines. Dried T 3 seeds of all transformants, and seeds of Kinmaze and EM10, were de-hulled. Except for line #1-1 and EM935, 10% of the outer seed layer was removed with a rice polisher (Kett, Tokyo, Japan). The unpolished seeds of line #1-1 and EM935, and the polished seeds of the other lines, were powdered in a coffee mill (National, Osaka, Japan) and filtered through 150 µm mesh. The filtered powder ( g) was separated into the soluble and insoluble fractions according to the above method. The combined supernatants were mixed with three volumes of methanol and centrifuged at g for 5 min at 20 C. The resulting precipitate was used for Sephacryl S-1000SF chromatography of soluble polyglucans in #1-1 and EM935. For SEM observations, X-ray diffraction and Sephacryl S- 1000SF chromatography of the insoluble polyglucans in #1-1, Kinmaze and EM10, the insoluble polyglucans were washed three times with 2% sodium dodecylsulphate (SDS), five times with distilled water and twice with 100% methanol, and then dried. Size separation of soluble and insoluble α-polyglucans by Sephacryl S-1000SF chromatography The size separation of soluble and insoluble polyglucans was carried out by the chromatographic method of Kubo et al. (1999). After chromatography, an aliquot of each fraction was taken for the measurement of carbohydrate content by the phenol sulphuric method. Analysis of the chain length profiles of Sephacryl S- 1000SF fractions After Sephacryl S-1000SF chromatography of soluble and insoluble polyglucans, peak fractions were collected (see Figure 4A,B), mixed with three volumes of methanol and kept on ice overnight. The solution was then centrifuged at g at 4 C for 10 min, and the precipitated polyglucans were washed with 5 ml of 100% methanol and dried. The polyglucans were debranched and their chain length distribution was determined by capillary electrophoresis as described above. X-Ray diffraction and SEM observations of starch granules These procedures were performed according to Fujita et al. (2003). Acknowledgements The authors thank Dr P. B. Francisco Jr. for reading the manuscript. References Baba, T. and Arai, Y. (1984) Structural characterization of amylopectin and intermediate material in amylomaize starch granules. Agric. Biol. Chem. 48, Blennow, A., Hansen, M., Schulz, A., Jørgensen, K., Donald, A.M. and Sanderson, J. (2004) The molecular deposition of transgenically modified starch in the starch granule as imaged by functional microscopy. J. Struct. Biol. 143, Boyer, C.D. and Preiss, J. 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