The Plant Journal (1997) 12(1), 143-153

Correlation between activities of starch debranching enzyme and (x-polyglucan structure in endosperms of sugary-1 mutants of rice

1 t , Yasunori Nakamura • , Aklko Kubo 2, Tomoyuki Shimamune 3, Toshiaki Matsuda 9, Kyuya Harada z and Hikaru Satoh 4 1National Institute of Agrobiological Resources, Kannondai, Tsukuba, Ibaraki 305, Japan, 2Department of Horticulture, Chiba University, Matsudo, Chiba 271, Japan, 3Department of Agriculture, Ibaraki University, Ami, Tsuchiura, Ibaraki 300-03, Japan, and 4Faculty of Agriculture, Kyushu University', Hakozaki, Higashi-ku, Fukuoka 812, Japan

Summary

The biochemical lesion of the sugary-1 mutation was examined in five different mutants of rice with varying phenotypes but with mutations at the same locus. The cells in the inner part of the endosperm of all mutants tested contained phytoglycogen instead of starch, while the cells located in the outer part of the endosperm tissue from some mutants were filled with numerous starch granules. The molecular size of phytoglycogen was mark- edly smaller than that of amylopectin as measured by Sephacryl S-1O00 chromatography. Analysis of the distri- bution of a-1,4 chain lengths revealed that in phytogly- cogen the number of A-chains dramatically increased, while long B chains with DP I> 37 remarkably decreased or were almost absent, which resulted in the disappear- ance of the cluster structure. The results suggest that changes in the balance of enzymic activities induced by the mutations brought about a drastic alteration in polyglucan structure and the shape of the polyglucan granule. The greater the extent of phytogiycogen regions in sul endo- sperm tissues became, the greater was the phytoglycogen content, and the greater the reduction in the activity of starch debranching enzyme, a type of enzyme referred to as R-enzyme (RE}, limit dextrinase or puUulanase. Immuno- blot analysis showed that the reduction in RE activity was due to a decrease in the amount of RE protein, and that the reduction in RE was specific since proteins of starch- branching enzymes I and Ila and ADP-glucose pyrophos- phorylase were not markedly affected by sul mutations. The proportion of starch region to the whole endosperm

Received 4 September 1996; revised 28 February 1997; accepted 5 March 1997. *For correspondence (fax +81 298 38 8347).

tissue of various sul mutants was correlated with the RE activity in these endosperms. The results strongly suggest that the reduction in RE activity is involved in the sul phenotype and that the enzyme plays an essential role in determining the fine structure of the amylopectin molecule.

Introduction

c~-l,6-glucosidic bonds in amylopectin, a major constituent of starch in a variety of plant tissues, are formed by starch- branching enzyme (BE) (EC 2.4.1.18). It is widely accepted that amylopectin is synthesized by the combined actions of BE and starch synthase (EC 2.4.1.21) which catalyzes the c~-1,4 chain elongation reaction. However, recent studies with sugary-1 (sul) mutants of maize (James et al., 1995; Pan and Nelson, 1984) and rice (Nakamura et al., 1992b,1996b), all show that amylopectin is replaced by phytoglycogen with more highly branched, water-soluble polysaccharide, strongly suggest that starch-debranching enzymes also play an essential role in determining amyl- opectin fine structure, since the activities of two types of starch-debranching enzymes, i.e. RE (EC 3.2.1.41) and isoamylase (EC 3.2.1.68), were selectively reduced in the sul mutants.

The biochemical lesion of the sul mutation has been reported by several laboratories (Nakamura, 1996). Pan and Nelson (1984) showed that the sul endosperm lacks the enzyme activity of one of the three isoforms of RE and possesses low activities of the other two RE isoforms. They also reported that the Km values for phytoglycogen for the latter two forms are three-fold higher in the mutant than those in the parent cultivar. These results demonstrate the combined effects of the loss or lowering of RE activities and the poor affinity for phytoglycogen in sul endosperm. It is not clear, however, whether the effects are specific to RE activities or not, because they did not assay the other enzymes involved in starch metabolism. Later, Dohlert et al. (1993) reported that the activities of the total amylase and c~-amylase as well as of RE are lower in sul kernels of maize than in normal cultivars.

Nakamura et al. (1992b,1996b) assayed the major enzymes of starch and sucrose metabolism in developing endosperms from sul mutants of rice and showed that the activity of RE specifically decreases in all sul mutants tested. The activities of starch-branching enzyme I (BEI), a major constituent of BE isozymes in rice endosperm

143

144 Yasunori Nakamura et al.

Starch debranching enzyme and amylopect in structure 145

(Yamanouchi and Nakamura, 1992), are significantly lower in the mutants than in the parent cultivars, while activities of the other BE isozymes, BEIla and BEIIb, are comparable in the mutants and their parent cultivars (Nakamura et aL, 1992b,1996b). However, the drop in RE activity is much more pronounced than that in BEI activity; hence, the ratio of RE activity to BEI activity is markedly lower in sul endosperm than in normal wild-type endosperm (Nakamura et al., 1996b).

Recently, James et aL (1995) isolated the gene SUI in maize endosperm by transposon tagging. It is highly possible that the gene encodes isoamylase because the deduced amino acid sequence from then cDNA product is highly similar to that of a bacterial isoamylase. However, the sequence greatly differs from that of a bacterial pullul- anase or RE from rice endosperm. Later, they found that while the SU1 product exhibits an isoamylase-type capa- city, it cannot attack pullulan (A.M. Myers, personal com- munication), indicating that the SU1 product is isoamylase, and not RE.

Mouille et al. (1996) isolated sugary-like mutants of Chlamydomonas sta7, where starch is replaced by glyco- gen-like polysaccharides and showed that an isoamylase- type debranching enzyme is selectively lacking.

All these results support the view that starch-debranch- ing enzymes are involved in the determination of amylopec- tin structure and that loss or reduction in the debranching activity results in the production of phytoglycogen at the expense of amylopectin synthesis.

However, it should be stressed that the possible function of RE in amylopectin biosynthesis in rice endosperm has been indicated only by indirect evidence. To elucidate the mechanism for the involvement of the enzyme in controlling amylopectin structure, more detailed data are needed. This paper presents experimental results obtained from endosperms of a variety of sul mutants of rice indicating a close relationship between the levels of RE activity and the extent of sugary phenotypes. The paper also deals with influences of sul mutations on the polyglu- can fine structures.

Results

Effects of sugary-1 mutations on the distribution of starch and phytoglycogen in developing rice endosperm

Endosperm from five different lines of sugary-I (sul) mutants of rice, i.e. EM-5, EM-41, EM-273, EM-914 and EM-

935, was used in this study. Although the parent cultivars of the former three and the latter two were Kinmaze and Taichung-65, respectively, it was confirmed that the sugary mutant genes of EM-41, EM-273, EM-914 and EM-935 are allelic to the sul gene of EM-5 (H. Satoh, unpublished data), which is localized on chromosome 8 (Yano et aL, 1984). While the degree of the sugary phenotype varied, all of these sul mutants of rice possess the common phenotype of a large amount of water-soluble (z-poly- saccharides termed phytoglycogen (Sumner and Somers, 1944) inside 'glassy' endosperm cells. When the median cross-section of the kernel of EM-5 was stained with iodine solution, only the outer layers of the endosperm tissue exhibited a blue-to-purple coloration, indicating the extent of accumulation of starch grains in these cells (Figure lb). The inner region of the kernel was not stained with iodine solution, demonstrating the replacement of starch by phy- toglycogen. The existence of an intermediate region of one or two layers of endosperm cells in EM-5 suggests that starch and phytoglycogen co-exist or some intermediate glucans between starch and phytoglycogen accumulate in such intervening cells. In EM-41, the starch region was dramatically reduced and confined to the few outermost layers of the endosperm cells (Figure lc). Only a part of the one or two outermost layers of endosperm cells of EM- 273 were stained with iodine solution (Figure ld), while almost all of the endosperm cells of EM-914 were devoid of starch granules (Figure lg). The extent of the region of endosperm cells containing phytoglycogen in EM-935 was between that of EM-5 and EM-41, and the intermediate region in EM-935 was significantly wider than that in EM- 5 (Figure lf).

Effects of sugary-1 mutations on (~-polyglucan structure in developing endosperms of rice

To examine and compare in detail the fine structure of ~- polyglucans stored in the endosperm of a variety of sul rice mutants and their parent cultivars, the chain length distribution of their isoamylolysates were analyzed using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). The c~- 1,4 chains of amylopectin from the parent cultivars, Kin- maze or Taichung-65, exhibited two major peaks (Figure 2a and e). The small peak corresponded to the long B-chains (DP /> 37) responsible for the regularly repeated cluster structure of amylopectin (Peat et al., 1952). This peak was greatly reduced in EM-5 (Figure 2b) and EM-935 (Figure 2g), and almost disappeared in EM-41, EM-273 and EM-914

Figure 1. Light micrographs of cross-sections through kernels at the mid-milky stages of sugary-1 mutants and their parent cultivars of rice. Sections were stained with iodine solution. (a) Kinmaze; (b) EM-5; (c) EM-41; (d) EM-273; (e) Taichung-65; (f) EM-914; (g) EM-935. The cross-sections were prepared from the middle part of the kernels, except in Taichung-65 where the section was taken from the upper part. For details, see Experimental procedures.

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Figure 2. HPAE-PAD analysis of the total (z-polysaccharides from various sugary-I mutants and their parent cultivars. The polyglucan samptes were debranched with P. amyloderamosa isoamylase and reduced with sodium borohydrate. The distribution of the reduced debranched (z-l,4-glucans with different chain lengths was analyzed as described in Experimental procedures. Numbers on the peaks indicate the DP values of the respective rz-l,4-glucans. (a) I<inmaze; (b) EM-5; (c) EM-41; (d) EM-273; (e) Taichung-65; (f) EM-914; (g)EM-935.

(Figure 2c, d and f, respectively). On the other hand, the amount of short chains w i th DP in the range of 4-10 markedly increased in these sul mutants. The results suggest that the sul mutat ion caused a drastic change of amylopect in into phytoglycogen- l ike polyglucans wi th

smaller side chains, lacking the cluster structure, and the degree of these changes varied depending on the sul l ines.

Hanashiro et aL (1996) recently reported that amylopec- t in side-chains can be classified into four groups: namely, fa wi th DP values 6-12, fb l wi th DP 13-24, fb 2 wi th DP

Starch debranching enzyme and amylopectin structure 147

Table 1. Distribution of (~-l,4-glucan chains in the total (x-polysaccharides in mature endosperms of various sugary-1 mutants of rice and their parent cultivars, as determined by HPAE-PAD analysis

Cultivar fa (DP~<12) fbl (13~<DP ~<24) fb2 (25~<DP~<36) fb3 (37~<DP)

Percentage of total peak area Kinmaze 27.77_+0.21 47.13_+0.30 10.86_+0.31 14.24-+0.25

EM-5 36.12_+0.12 43.16_+0.38 12.42_+0.17 8.30_+0.27 EM-41 44.46_+0.62 41.40_+0.38 9,88_+0.37 4.26_+0.59 EM-273 52.10_+0.76 37.12_+0.42 8,59_+0.23 2.19_+0.31

Taichung-65 28.63_+2.91 44.95_+1.45 11.14_+0.89 15.28_+0.59 EM-914 45.63_+0.96 42.07_+0.20 10,29_+0.42 2.00+-0.46 EM-935 40.09+_0.90 42.01 -+0.56 11.66-+0.25 6.24_+1.12

The (~-1,4 chains of (~-polyglucans were classified into four groups according to Hanashiro et al. (1996). The peak area of each glucan chain was calculated, and the sums of the peak area corresponding to fa, fbl, fb2 and fb3 groups, respectively, were expressed as percentages of the total peak area. Each value represents the mean + SD of three replicate analyses. Figure 2 shows the data from such a representative analysis.

25-36, and fb3 with DP t> 37; these fractions presumably corresponding to A, B1, B2, B3 and longer chains, respectively. Table 1 presents the effects of the sul mutation on the chain-length distribution of c~-polygtucans based on this classification, except that short chains with DP smaller than 6 were also included in the fa fraction. It is apparent that short chains with DP ~< 12 dramatically increased and long B chains with DP ~> 37 conversely decreased in (~-polyglucans from sul mutants. The extent of these changes in sul mutants were approximately related to the area of the phytoglycogen region, namely the magnitude of the sul phenotype. It is also shown that the c(-1,4 chains with intermediate sizes of 13 <~ DP

36 (B 1 and B2 chains) were only somewhat decreased by the mutations. The results indicate that the highly organized cluster structure of amylopectin was destroyed in the sul polysaccharides.

To test whether the su 1 mutations affect the molecular size of polyglucans in addition to the distribution of side- chains, Sephacryl S-1000 SF (Pharmacia Biotech) gel filtra- tion chromatography was used, since the size of amylopec- tin present in plant storage organs has been reported to

7 9 rangefrom 10 to 10 ,whilethat of glycogen is ofthe order of 107 (Ball etaL, 1996; Manners, 1989; Manners and Sturgeon, 1982). As shown in Figure 3, the major peak of amylopectin eluted from the column in the range of fractions 8-14, while phytoglycogen was detected in fractions 16-28, indicating that the molecular size of phytoglycogen is markedly smaller than that of amylopectin. Judging from the high ~,rnax values giving the blue colors, fractions containing most, if not all, of the amylose were seen from 19-27, although almost all these fractions alsocontained phytoglycogen. The peakfrac- tions of 30-34 included free sugars composed primarily of sucrose (data not shown). Based on these observations, we concluded that the amylopectin, phytoglycogen plus amyl- ose, and free sugars were at fractions 8-14, 19-28, and 30- 34, respectively. Table 2 indicates that the decrease in the

amylopectin fraction was compensated by the increase in the phytoglycogen plus amylose fraction. The amount of amylose actually decreased in the sul mutants except for EM-935 and EM-5, since the Xmax value and the intensity of the absorbance at the respective ~max were lower in the mutants than those in the parent cultivars (Table 2).

Effects ofsugary-1 mutations on RE activities in developing rice endosperms

Figure 4 illustrates the changes in activities of RE in developing rice endosperms as influenced by the sul mutation. All the endosperm RE migrated to the same position on a native polyacrylamide gel, distinct from other amylolytic enzymes, almost all of which migrated at lower rates than RE. Each RE band was visualized as a blue band, while amylase bands were white-yellow. The intensity of the RE band significantly decreased in EM-5 compared with Kinmaze and it was apparent that the other sul mutants EM-41, EM-273, EM-914 and EM-935 were more significantly deficient in RE activity. Intensities of many, if not all, amylase bands were not significantly affected by the sugary mutations.

The levels of RE activities in maturing endosperm were directly measured using crude extracts as the enzyme source and pullulan as substrate. Table 3 shows that the activities of RE in EM-5, EM-41, EM-273, EM-914 and EM- 935 were 38, 28, 22, 15 and 30%, respectively, of those of their parent cultivars. These results are in agreement with those obtained from the native-PAGE/activity staining of RE (Figure 4).

Effects of sugary-1 mutations on the amounts of enzymes involved in starch biosynthesis in developing rice endosperms

It is important to determine whether the decrease in the RE activity in sul mutants of rice is due to the decrease in

148 Yasunori Nakamura et al.

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Figure 3. Separation of amylopectin, phytoglycogen and amylose by Sephacryl S-lO00 chromatography. Samples of the total (x-glucans were prepared from mature endosperms of sugary-1 mutants and their parent cultivars and fractionated as described in Experimental procedures. After chromatography, the content of the carbohydrate in each fraction (o) was determined by the phenolic sulfuric method. 0.8 ml each of the fractions was added to 0.2 ml of 0.1% 12/1% KI and the absorption spectrum of the iodine-polyglucan complex was measured and the 3,max determined (illustrated as a fine line), although no ~,ma× values were obtained in EM-273 and EM-914. (a) Kinmaze; (b) EM-5; (c) EM-41; (d) EM-273; (e) Taichung-65; (f) EM-914; (g) EM-935.

Starch debranching enzyme and amylopectin structure 149

Table 2. Percentage composition of the amylopectin fraction (I), the phytoglycogen plus amylose fraction (11), and the free sugar fraction (Ill) separated by Sephacryl S-IO00 chromatography in mature endosperms of various sugary-1 mutants of rice and their parent cultivars

Cultivar I (%) II (%) III (%) II/I ~,rnax* Absorbance**

Kinmaze 46.46_+ 1 . 5 9 31.99-+0.43 2.61 _+0.88 0.69 613 0.723 EM-5 31.26_+0.41 46.73_+0.61 6.00_+0.40 1.49 607 0.789 E M-41 17.02 -+ 1.18 53.71 _+ 1.69 15.46 +_ 1.35 3.16 599 0.495 EM-273 10.50_+ 1.03 59.87_+ 1 . 4 8 16.83_+0.17 5.70 575 0.200

Taichung-65 38.91 _+0.66 41.00-+0.35 5.01 -+0.36 1.05 614 0.724 EM-914 1.58_+0.08 72.34+0.08 14.41 _+1.17 45.8 452 6.176 E M-935 15.61 -+ O. 67 55.73 _+ 0.88 14.86 + 0.81 3.57 614 0.728

The amylopectin (I), phytoglycogen plus amylose fraction (11), and free sugar fractions (Ill) corresponded to fractions 8-14, 19-28, and 30- 34, respectively, in the Sephacryl S-lO00 chromatogram, as shown in Figure 3. The amounts of carbohydrates in fractions I, II and III were expressed as percentages of total carbohydrates in the Sephacryl S-IO00 chromatogram. The experiments were the same as in Figure 3. *The ~'max values were those measured using fraction 26 in each chromatogram, although the values of the shoulder peak were used for EM-273 and EM-914. **The value corresponding to the absorbance at the respective ~'max.

Table 3. Activities of R-enzyme in maturing endosperms of various sugary-1 mutants of rice and their parent cultivars

Cutivar or line R-enzyme activity (nmol min -1 per endosperm) (%)

Kinmaze 68.3 - 6.0 (100) EM-5 26.0 - 3.8 (38) EM-41 19.3 -+ 3.3 (28) EM-273 15.0 - 1.6 (22)

Taichung-65 84.5 _+18.6 (100) EM-914 12.7 _+ 3.8 (15) EM-935 25.2 _+ 8.2 (30)

Each value represents the mean +- SD of three separate experiments. The enzyme activity obtained in each experiment was the mean of three replicate incubations.

Figure 4. Native-PAGE/activity staining of RE in developing endosperm of sugary-1 mutants and their parent cultivars. The volumes of the crude enzyme extract applied were as follows: upper panel: lanes 1-4, 3.3 Id; lanes 5-8, 6.7 I11; lanes 9-12, 13 td. Lower panel: lanes 1-5, 3.3 ~1; lanes 6-10, 6.7 ~1. The arrow indicates the band corresponding to RE. For details, see Experimental procedures.

the enzyme amount, or due to modif ication of the amino

acid sequence of RE induced by the sul mutation. Western

blot analysis using a polyclonal ant ibody raised against

the purified RE from developing rice endosperm indicates

that EM-5 had a significantly reduced amount of RE protein

compared with its parent cultivar Kinmaze (Figure 5). The

RE amounts dropped more severely in EM-41, EM-273,

EM-914 and EM-935. The results suggest that the sul mutation inhibited expression of the RE gene in developing

endosperm of rice. Figure 5 also shows that the sul mutations did not

cause any strong reduction in the amounts of ADP-glucose

pyrophosphorylase (ADPP), starch-branching enzyme I

(BEI) and starch-branching enzyme Ila (BEIla) in immuno-

blot experiments, except that EM-914 contained a some-

what lower level of BEI and BEIla proteins. In summary,

these results indicate that the sul mutation selectively

brought about a decrease in the amount of RE protein.

150 Yasunori Nakamura et al.

Figure 5. Western blot analysis. (a) RE; (b) BEI (starch-branching enzyme I) (Nakamura et aL, 1992a); (c) BEIla (starch-branching enzyme Ila) (Nakamura et al., 1992a); (d) ADPP (ADP-glucose pyrophosphorylase). Each lane in panels (a) and (d) and in panels (b) and (c) contained 6.7 and 1.3 IJI, respectively, of the crude extract prepared from various kinds of developing rice endosperms, as indicated in the figure, and subjected to SDS-PAGE. tmmunoblot analyses were performed using the antisera raised against the purified RE from developing rice endosperm (Nakamura et al., 1996b), rice endosperm BEI (Nakamura et al., 1992a), rice endosperm BElla (Nakamura et al., 1992a) and the isoform $5 of rice endosperm ADPP (Nakamura and Kawaguchi, 1992). For details, see Experimental procedures.

Discussion

The present study shows that the su l mutation greatly influences not only the composition but also the polyglucan structure in developing rice endosperm.

Figure 1 demonstrates that the endosperm cells of several su l mutants of rice are composed of ordinary cells con- taining a large amount of starch inside the amyloplasts, and altered cells where phytoglycogen is seemingly synthesized at the expense of normal starch (amylopecti n). These altered cells are located in the central part of the endosperm. The magnitude of changes in the distribution of c~-1,4 chains in polyglucan (the fine structure of the polyglucans) and the proportion of phytoglycogen to amylopectin as measured by HPAE-PAD (Figure 2) and Sephacryl S-1000 chromato- graphy (Figure 3), are related to the ratio of the area of the phytoglycogen region tothat of the starch region (Figure 1). The results strongly suggest that some biochemical changes such as the balance of activities involved in starch biosyn- thesis can produce a remarkable change in starch structure.

We previously proposed that a deficiency in RE activity is a biochemical lesion of su l mutation of rice endosperm, since the RE activity is selectively reduced in su 1endosperm,

while the activities of ADP-glucose pyrophosphorylase, sol- uble starch synthase, c~-glucan phosphorylase, c~-amylase, 13-amylase, UDP-glucose pyrophosphorylase and sucrose synthase are basically unaffected by the sul mutations, and that of BE is slightly decreased (Nakamura et al.,

1992b,1996b). The present results shown in Figures 4 and 5 and Table 3 further indicate that the extent of the decrease in RE activity is basically proportional to the magnitude of the sugary phenotype in rice endosperm (Figures 1-3 and Tables 1 and 2). Immunoblot experiments (Figure 5)indicate that the decrease in RE activity is due to a reduction in the amount of RE. Figure 5 also shows that the RE amount is specifically low in su 1mutants, becausethe protein amounts of ADP-glucose pyrophosphorylase, BEI and BEIla are basic- ally unaffected by the su I mutation in rice endosperm.

In an attempt to clarify whether the decrease in RE activity correlates with the sul phenotype in rice endosperm, the relative volume of the phytoglycogen region to the starch region was calculated. If the rice endosperm tissue is a com- plete sphere in shape, the volume of the region is propor- tional to (area) 3/2 of the corresponding region. On the other hand, if the endosperm tissue forms a pillar, the volume of the region is proportional to the area of the region. The ratio of the volume of the phytoglycogen region to the starch region may actually be between the above volumes calcu- lated from the two assumptions. Figure 6 compares these volumes with the RE activities detected in endosperms from a variety of su l mutants of rice. It can be seen that the activities of RE in endosperms decrease with the decrease in the volume of the starch region, and the activity of RE is lowest in EM-914 where the starch region is barely visible, indicating the close correlation between the magnitude of su l phenotype and the level of RE activity. Since the activities of RE are more highly correlated with the volume of the starch region than with that of the starch region plus the intermediate region, it is possible that the intermediate region, if not the whole, contains phytoglycogen-like poly- saccharides. In conclusion, the present results support the idea that the su l phenotype in rice endosperm is related to the deficiency in RE activity.

Pan and Nelson (1984) reported that one of the three iso- forms of RE is deficient in the endosperm of su l of maize, while James etal. (1995) demonstrated by gene tagging that the SU1 gene encodes isoamylase in maize. Dohlert et al.

(1993) showed that the decrease in RE activity is not specific, since the activities of RE and the other hydrolytic enzymes such as amylases are significantly reduced in maize sul

endosperm at similar levels. It was not proven whether or not the lack of the RE activity is due to the direct lesion of the gene for RE or to indirect results brought about by biochem-

152 Yasunori Nakamura et al.

buffer (pH 7.2) for 2 h at 4°C, and post-fixed in 1% (v/v) aqueous solution of osmium tetroxide for 12 h at 4°C. The cross-sections (0.5-1.0 pm thick) were prepared from the middle part of the kernels with a glass knife on an Ultra microtome. These specimens were dehydrated in a graded ethanol series and embedded in Spurr's resin (TAAB) (Spurr, 1969). The resin was extracted with NaOH- saturated ethanol. Sections were stained with I/KI solution (Jensen, 1962) and observed under a light microscope (Olympus, VANOX AHD-LB).

Preparation of enzymes

The fol lowing procedures were carried out at 0-4°C, unless other- wise stated. Samples of 10 maturing rice endosperms at the late- milky stage were separated from embryo and pericarp and homo- genized in a pre-chilled mortar and pestle with 1 ml of chilled buffer solution containing 50 mM imidazole-HCI (pH 7.4), 8 mM MgCI2, 50 mM 2-mercaptoethanol and 12.5% (v/v) glycerol. The homogenate was centrifuged at 15 000 gfor 5 min, and the supernatant was used as the enzyme preparation.

Native PA GE/activity staining of RE

The method of Davis (1964) with modifications was used in this study. Native slab polyacrylamide gels (6 x 9 × 0.1 cm) consisting of 7.5% (w/v) acrylamide and 0.15% (w/v) potato tuber amylopectin (Sigma) were used for the resolving gel and 3.3% (w/v) acrylamide for the stacking gel. Electrophoresis was performed at a constant current of 10 mA for 2.5 h at 4°C. For detection of the RE activity, the gel was rinsed twice with 20 ml of 50 mM Na-acetate (pH 5.4), 50 mM 2-mercaptoethanol and 50 mM CaCI 2 at room temperature and then imbibed at 37°C for 2.5 h with 20 ml of the same buffer solution. RE activity was visualized by incubating the gel in 30 ml of 0.1% (w/v) 12 in 1% (w/v) KI solution.

Western blotting

SDS-PAGE was performed on a resolving gel (6 x 9 x 0.1 cm) of 10% (w/v) acrylamide in the absence of urea fol lowing the method of Laemmli (1970). The protein bands in the gel were blotted onto a nitrocellulose membrane (Schleicher & Schuell) by Western blot- ting, and the filter was incubated with a rabbit antiserum containing polyclonal antibodies raised against the purified RE from develop- ing rice endosperm (Nakamura et aL, 1996a), the purified BEI from developing rice endosperm (Nakamura et al., 1992a), the purified BEIla from developing rice endosperm Ila (Nakamura etaL, 1992a), or the purified ADP-glucose pyrophosphorylase isoform $5 from developing rice endosperm (Nakamura and Kawaguchi, 1992). The immunoreactive bands were detected by the method of Towbin etal. (1979), as described previously (Nakamura etal., 1992a).

Assay of RE

The assay mixture for RE consisted of 50 mM Mes-NaOH (pH 6.4), 2 mg pullulan (Sigma), and the enzyme preparation in a total volume of 0.2 ml. The reaction was run for 20 min at 40°C and terminated in boiling water for 1 min. RE activity was determined by measuring the absorbance at 520 nm as an increase in reducing sugars on the basis of the methods of Nelson (1944) and Somogyi (1952), using maltotriose as standard. The enzyme assay was performed in the range where the activity linearly increased with enzyme concen- trations.

Analysis of ct-polyglucan structure

Determination of the distribution of a-l,4-glucan chain length in ct-polysaccharides by HPAE-PAD. The embryo and pericarp were removed from 10 dehulled average-sized grains. The endosperms were ground in a mortar and pestle and 5 mg of the resulting powder was suspended in 5 ml of methanol and boiled for 10 min. The homogenate was centrifuged at 2500 g for 10 min. The precipitated polyglucan fraction was washed twice with 5 ml of 90% (v/v) methanol, suspended in 5 ml of distilled water and then boiled for 60 min. An aliquot (1.0 ml) of the gelatinized polyglucan sample was added to 50 ~1 of 600 mM Na-acetate buffer (pH 4.4) and 10 t~l of 2% (w/v) NaN 3, and hydrolyzed by adding 10 ~1 of Pseudomonas amyloderamosa isoamylase (1400 units, Seikagaku Corporation, Tokyo, Japan) at 40°C for 24 h. The hydroxyl groups of the debranched glucans were reduced with 25 mg sodium borohyd- ride under alkaline pH condition for 20 h bythe method of Nagamine and Komae (1996).

The preparation was dried in vacuo at room temperature. The reduced isoamylolysate sample was dissolved in 100 ~1 1 M NaOH for 60 min and diluted with 900 I11 distilled water. An aliquot (25 I~1) of the preparation was injected into a Dionex BioLC (Model DX-500, Dionex, CA, USA) equipped with a pulsed amperometric detector and a CarboPac PA-1 column (4 mm x 25 cm). The size fractionation of(~-l,4-glucans was performed with a linear gradient of Na-acetate (25-250 mM) in 0.1 M NaOH at a f low rate of 1 ml min -1.

Molecular size separation of (~-polyglucans by Sephacryl S-1000 chromatography. The endosperms were ground in a chilled mortar and pestle, and 40 mg of the powder was suspended with 4 ml of 1 M NaOH. After 30 min at room temperature, 4 ml of 0.1 M NaOH was added to the sample suspension, and then centrifuged at 2000 g fo r 5 min at 25°C. Four ml of the supernatant were applied onto a Sephacryl S-1000 (Pharmacia Biotech, Uppsala, Sweden) column (2.0 cm diameter; 60 cm long) which had been equilibrated with 0.1 M NaOH containing 0.2% (w/v) NaCI. The sample was eluted with the same solution at a f low rate of about 18.3 ml h -1 at room temperature. Fractions were taken at 200 drop-intervals (equivalent to 6.6 ml).

For measurement of the total carbohydrate content by the phenolic sulfuric method (Hodge and Hofreiter, 1962), 100 ~1 of each sample was taken and added successively to 200 I~1 distilled water, 10 I~1 80% (v/v) phenol and 800 IJI sulfuric acid. The absorbance of the mixture was measured at 490 nm.

For determination of a ~,max value of the polysaccharide-I 2 complex, 800 ~1 of each sample was mixed with 200 I11 distilled water, 100 ~1 1 M HCI and 200 ~1 0.1% 12/0.01% KI, and the absorption spectrum of the resulting solution was obtained in the 450-700 nm range.

Acknowledgments

We thank Ms Yumiko Inaba and Kazuko Kimura for their technical assistance and Dr P.B. Francisco Jr for reading the manuscript. We also thank the staff of Supporting Service of NIAR for their help in growing the plant materials. The work was supported by grants from Special Coordination Funds for Promoting Science and Technology, the Enhancement of Center-of-Excellence (COE) Program, Japan, and from the Ministry of Agriculture, Forestry and Fisheries, Japan.

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