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Distribution of storage proteins in low-glutelin rice seed determined using a fluorescent antibody
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 96, No. 5, 467–473. 2003
Distribution of Storage Proteins in Low-Glutelin Rice Seed Determined Using a Fluorescent Antibody SACHIKO FURUKAWA,1* TOMOCHIKA MIZUMA,1 YOSHIFUMI KIYOKAWA,1 TAKEHIRO MASUMURA,2,3 KUNISUKE TANAKA,2,3 AND YOSHINORI WAKAI1 Kizakura Sake Brewing Co. Ltd., 53 Kajiwara-cho, Shimomisu, Yoko-ohji, Fushimi-ku, Kyoto 612-8242, Japan,1 Laboratory of Genetic Engineering, Faculty of Agriculture, Kyoto Prefectural University, Shimogamo, Kyoto 606-8522, Japan,2 and Kyoto Prefectural Institute of Agricultural Biotechnology, Seika, Kyoto 619-0244, Japan3 Received 23 April 2003/Accepted 25 August 2003
To compare the distribution of storage proteins in low-glutelin rice seed with that in other cultivars having normal protein compositions, immunofluorescence labeling with specific antibodies was applied to visualize the distribution of storage proteins in endosperm tissues. The endosperm tissues from five cultivars were reacted with anti-prolamin and anti-glutelin antibodies, and then observed by light microscopy and confocal laser scanning microscopy (CLSM). In low-glutelin rice, using microscopic analysis, a large proportion of storage proteins was observed in the endosperm tissue of 70% polished rice. To determine the localization of two types of protein bodies in endosperm tissues, images of the distribution of the type I protein body (PB-I) and the type II protein body (PB-II) were obtained by CLSM. The CLSM images showed that, in low-glutelin rice, prolamin which accumulates in PB-I remains in the center of 70% polished rice grains despite the elimination of 30% of the outer layer of brown rice grains. However, the other cultivars mostly contained glutelin which accumulates in PB-II and is distributed throughout the endosperm tissues. This shows that low-glutelin rice differs from the other cultivars not only in the major storage protein composition but also in the distribution of storage proteins in endosperm tissues. [Key words: confocal laser scanning microscopy, protein body, fluorescent antibody technique, low-glutelin rice, sake brewing]
Observation by confocal laser scanning microscopy (CLSM) provides images with high depth and high resolution with a minimum of background noise (7). Positional relationship can be determined in the area of interest by differential staining (7). Several studies have been conducted on the behavior of rice protein bodies in the sake brewing process (8–11) and the morphology of rice grains as a raw material in sake brewing (7, 12, 13). However, to our knowledge, there has been no published study comparing the distribution of storage proteins in the endosperm tissues of rice used for brewing sake using a fluorescent antibody technique. For lowglutelin rice, of which the content of glutelin is reduced, we previously determined the effect of the composition of the rice protein on the sake brewing process, and indicated the possibility that prolamin is highly related to the dissolution of the raw material rice grain (13, 14). Therefore, we consider it would be interesting to investigate the distribution of storage proteins in the raw material rice grain and determine the effect on the sake brewing process. To visualize the distribution of rice seed storage proteins, we performed double immunofluorescence labeling with specific antibodies against prolamin and glutelin individually, and observed the results using CLSM. In this paper, we
In rice endosperm cells, seed storage proteins (proteins stored in seed) accumulate in two types of granules called protein bodies. It has been shown that the type I protein body (PB-I) stores prolamin as an alcohol-soluble protein, whereas the type II protein body (PB-II) mainly contains glutelin as an alkaline (and/or acid)-soluble protein (1, 2). PB-I cannot be digested by the human digestive system, whereas PB-II is digestible (3–5). Generally, rice protein bodies (japonica rice) consist of 20–30% PB-I and 70–80% PB-II. Rice is a staple food in Japan, and often used as a raw material for brewing Japanese sake. The function of rice grains in the sake brewing process is as a growth medium for yeast such as “kakemai” or koji as “rice-koji”. Free amino acids and peptides liberated from rice seed proteins are known to play an important role in enhancing the taste of sake (6). Rice grains containing small amounts of protein are suitable for brewing sake because surplus amino acids spoil the taste. Therefore, analytical information on rice seed proteins is indispensable to selecting rice cultivars as raw materials in sake brewing. * Corresponding author. e-mail: [email protected] phone: +81-(0)75-611-4101 fax: +81-(0)75-622-3510 467
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TABLE 1. The list of rice cultivars used in this study Crude protein contents (g/100 g) Protein composition of 70% polished rice (%) Brown rice 70% polished rice PB-I PB-II Others LGC1 * 7.2 5.1 41.4 36.7 21.9 Tashu-kei 1001 * 7.6 5.0 39.4 38.5 22.1 Hyogokitanishiki * 8.3 5.2 20.4 59.1 20.5 Yamadanishiki Hyogo 7.1 4.5 22.5 58.0 19.5 Nipponbare Shiga 7.9 5.2 22.3 58.7 19.0 * The National Agricultural Research Center for the Western Region. Protein composition was analyzed according to Kizaki et al. (8). PB-I represents the type I protein body and PB-II represents the type II protein body. Rice cultivars
Locality
report the different distributions of storage proteins in the rice grains of low-glutelin rice and other cultivars used for brewing sake.
labeled samples. The processing images for the localization of PB-I (prolamin) and PB-II (glutelin) in endosperm cells were obtained separately and merged.
MATERIALS AND METHODS
RESULTS
Materials Table 1 shows the rice (Oryza sativa L.) cultivars used in this study, which were harvested in 2000. Three cultivars listed in Table 1, i.e., LGC1, Tashu-kei 1001 and Hyogokitanishiki, were from the National Agricultural Research Center for the Western Region. LGC1, which contains small amounts of glutelin and large amounts of prolamin, is a low-glutelin rice used in the diet of patients with kidney disease (15). Tashu-kei 1001 was cultivated as a hybrid of LGC1 and Hyogokitanishiki to be used for sake brewing, and genetically has the protein composition of LGC1 (13, 14). To obtain polished rice for sake brewing, brown rice was polished to 70% of its original weight using a pearling mill (the graintesting mill; Satake, Higashi-Hiroshima). SDS–PAGE of brown rice and 70% polished rice proteins The rice seed storage proteins were extracted from the grains of brown rice and 70% polished rice (polished to 70% of its original weight) according to the methods of Kizaki et al. (8), and separated by SDS–PAGE using 15% acrylamide gels. The gels were stained with Coomassie Brilliant Blue R-250 (CBB R-250), and the protein composition was analyzed using a densitometer (Densitograph AE-6920M; ATTO, Tokyo). Preparation of cross sections A single grain of brown rice or 70% rice was cross-sectioned with a laser blade. The cross section was attached to a glass slide for observation under a fluorescence microscope. Then, thin cross sections of 200 mm were cut with a sliding microtome (HM400R; Microm, Walldolf, Germany). Observations by CLSM Immunofluorescence labeling using specific antibodies against prolamin and glutelin was conducted as follows. Cross sections of rice grains were fixed in 4.0% (w/v) paraformaldehyde and 0.5% (v/v) glutaraldehyde in phosphate buffer (PBS, pH 7.2), then blocked in 1.0% (w/v) BSA in TBST (0.05% [w/v] Tween 20 and 150 mM NaCl in 10 mM Tris–HCl [pH 8.0]) at room temperature for 1 h. Then, the fixed specimens were washed twice with TBST for 10 min at room temperature. After fixation and washing, tissue sections were reacted with a specific antibody from rabbit against the 13 kDa prolamin and from mouse against the 23 kDa glutelin for 1 h at 37°C. Reacted tissue sections were washed three times with TBST for 10 min, then, the secondary antibody was subsequently reacted with a FluoroLink™ Cy™2 (FITC)-labeled goat anti-rabbit IgG (Amersham Biosciences, Tokyo) and a FluoroLink™ Cy™3 (Rhodamine)-labeled goat anti-mouse IgG (Amersham Biosciences) for 1 h at 37°C. After washing with TBST as described above, tissue sections were observed using CLSM. A CLSM system (MRC-1024; Bio-Rad Laboratories, Hercules, CA, USA) was used for observing double immunofluorescence
Comparison of protein composition among rice cultivars Table 1 shows the crude protein contents of the brown rice and 70% polished rice used in this study. The crude protein content of Yamadanishiki 70% polished rice is 4.5 (g/100 g) which is lower than that of the other cultivars for which the crude protein content is approximately 5.0 (g/100 g). The storage proteins of brown rice and 70% polished rice were separated by SDS–PAGE. Figure 1 shows the SDS– PAGE protein band patterns of brown rice and 70% polished rice (cv. Hyogokitanishiki, LGC1 and Tashu-kei 1001). A comparison of these three cultivars showed that the protein band patterns of the brown rice and 70% polished rice exhibited distinct features. More precisely, in LGC1 and Tashu-kei 1001, the prolamin (10 kDa, 13 kDa, and 16 kDa polypeptides) content increased while the glutelin (22–23 kDa and 37–39 kDa polypeptides) content decreased compared to in Hyogokitanishiki. The crude protein content of
FIG. 1. SDS–PAGE protein band patterns of brown rice and 70% polished rice grains. Lane 1, Hyogokitanishiki; 2, LGC1; 3, Tashu-kei 1001; 4, Hyogokitanishiki; 5, LGC1; 6, Tashu-kei 1001. Lanes 1 to 3, Brown rice; lanes 4 to 6, 70% polished rice.
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Hyogokitanishiki brown rice was 8.3 (g/100 g), which is higher than those of LGC1 and Tashu-kei 1001; however, there was little difference among these cultivars in the total protein content of 70% polished rice, as shown in Table 1. The protein band patterns of Yamadanishiki and Nipponbare were similar to that of Hyogokitanishiki (data not shown). The protein compositions of the 70% polished rice were determined from the SDS–PAGE protein band patterns of these five cultivars, and are shown in Table 1. In LGC1 and Tashu-kei 1001, the protein composition consisted of higher level of PB-I and lower level of PB-II in comparison with the others. Thus, low-glutelin rice differed from Hyogokitanishiki and Yamadanishiki rice suitable for sake brewing and Nipponbare rice used for general consumption in the major protein composition. Distribution of storage proteins in low-glutelin rice endosperm To examine the distribution of storage proteins in low-glutelin rice, 70% polished rice was observed by light microscopy (BH-2; Olympus, Tokyo). Figure 2 shows the micrographs of a cross section of the 70% polished rice endosperm of Tashu-kei 1001 (Fig. 2A) and Hyogokitanishiki (Fig. 2B). In Fig. 2A, it is possible to clearly distinguish the area in which the storage proteins are stained blue with CBB (indicated by arrows as PB), and the starch granules observed as white. A large proportion of storage proteins could be observed in the outer layer of the 70% polished rice grain (the upper side of Fig. 2A). However, in Fig. 2B, the storage proteins stained with CBB could be observed to be spread thinly over the entire. Generally, PB-II is mostly distributed throughout the endosperm tissues, whereas PB-I is mostly confined to the outer layer of the grain (16, 17). Therefore, in the sake brewing process, polishing is useful for eliminating storage proteins from the outer layer of grains as is well known. That is, 70% polished rice consists almost completely of the starchy endosperm tissues without the aleurone or subaleurone layer because of the elimination of 30% of the outer layer of brown rice grains. However, in the lowglutelin rice Tashu-kei 1001, a large amount of storage proteins could be observed in the 70% polished rice as shown in Fig. 2A. This suggests that the distribution of storage proteins in the low-glutelin rice endosperm differs fundamentally from that of the other rice cultivars as shown in Fig. 2B. Distribution of two types of protein bodies in rice endosperm using CLSM To visualize the distribution of rice seed storage proteins, we used CLSM. Figure 3 shows CLSM cross-sectional images of 70% polished rice grains (cv. LGC1) treated with double fluorescence labeling using the anti-prolamin and anti-glutelin antibodies. Figure 3A–C is images of the storage proteins from the same rice grains. The distribution of prolamin (green) and glutelin (red) is displayed separately in Fig. 3A and 3B, but the two images are merged in Fig. 3C, thus the overlapping regions appear yellow. To determine the extent of nonspecific fluorescence generated by the antibody reaction, the fluorescence produced using only the primary or secondary antibody was detected. Figures 4A and 5A show sample results which indicate that the level of nonspecific fluorescence generated
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by the antibody reaction was very low and did not affect the detection of fluorescence with respect to PB-I and PB-II. The distribution of storage proteins in the endosperm tissues of steamed rice and koji was previously reported by Kojima et al. (7). However, in this study, Fig. 3 shows the distribution of prolamin and glutelin in a rice grain. The distribution of the two types of protein bodies in brown rice Figure 4 shows the distributions of the two types of protein bodies in brown rice. Half of the grains were compared between the five cultivars shown in Table 1 using the CLSM merged images. Figure 4A, as the control image, reveals that the level of nonspecific fluorescence generated by the secondary antibody reaction to detect prolamin is very weak. In the other control images, the level of nonspecific fluorescence generated by the secondary antibody reaction is similar to that in Fig. 4A (data not shown). This means that the nonspecific fluorescence slightly affects the fluorescence generated due to the reaction of the antiprolamin and anti-glutelin antibodies under the experimental conditions described above. The merged images of the double fluorescence labeling with the anti-prolamin and anti-glutelin antibodies are as follows: LGC1 (Fig. 4B), Tashu-kei 1001 (Fig. 4C), Hyogokitanishiki (Fig. 4D), Yamadanishiki (Fig. 4E), Nipponbare (Fig. 4F). The storage proteins of brown rice, shown in yellow, are distributed in the outer layer of grains in all rice cultivars in Fig. 4. Notably, Fig. 4B, 4E and 4F clarify that the storage proteins (PB-I and PB-II) are located close to the cell walls in the endosperm cells, shown as yellow dots (inside the region surrounded by the dotted line). These results indicate that the storage proteins are mostly localized close to the cell walls in endosperm tissues, which surround starch granules (13, 16). In addition, Fig. 4 shows that the brown rice grains of the five cultivars appear to differ slightly in the distribution of storage proteins. That is, the proteins are mostly distributed on the outer layer of the grains. The distribution of the two types of protein bodies in 70% polished rice Because high polished rice is used as a raw material for sake brewing, investigation of the distribution of the two types of protein bodies in 70% polished rice is more important than that in brown rice. Figure 5 shows the distribution of the two types of protein bodies in 70% polished rice of all the cultivars used in this study. Figure 5B to 5F represents the same cultivars as represented in Fig. 4B to 4F. Figure 5A is the control image similar to that in Fig. 4A, and reveals that the nonspecific fluorescence generated in double staining has a little effect on the fluorescence obtained with anti-prolamin (PB-I) and anti-glutelin (PB-II) in 70% polished rice. In 70% polished rice, storage proteins are distributed throughout the endosperm tissues of every cultivars as shown in Fig. 5. It is well known that starch granules generally occupy most of the rice endosperm. However, these results show that the storage proteins are also important as major components even in the 70% polished rice. In this study, based on the size of a rice grain, the 30% eliminated by polishing represents about 500 mm from the outer layer of the brown rice grain. Therefore, most of the area shown in Fig. 4 does not exist in Fig. 5 because of
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FIG. 2. Light microscopic images of 70% polished rice endosperm of low-glutelin rice. (A) Tashu-kei 1001; (B) Hyogokitanishiki. Storage proteins are stained blue with CBB (arrows indicate PB). The starch granules appear as white spheres. The images show the outer layer of a 70% polished rice grain (upper), and close to the center of a 70% polished rice grain (lower). PB represents protein bodies. The schematic shown below the images demonstrates the observation position (side of cross section of 70% polished rice grains).
FIG. 3. Double fluorescence labeling of brown rice and 70% polished rice grains using CLSM. (A) Anti-prolamin antibody to determine distribution of PB-I; (B) anti-glutelin antibody to determine distribution of PB-II; (C) merged image. The distribution of PB-I (prolamin) and PB-II (glutelin) in the rice grains (cultivar, LGC1) was determined by fluorescence labeling. In Fig. 2A, the green signal is due to a reaction with a FluoroLink™ Cy™2 (FITC)-labeled goat anti-rabbit IgG and in Fig. 2B, the red signal to a reaction with a FluoroLink ™ Cy™3 (Rhodamine)-labeled goat anti-mouse IgG, which indicate the distribution of PB-I and PB-II, respectively. Yellow indicates the overlapping regions. Arrows indicate the outline of the 70% polished rice grain. The schematic shown below the images demonstrates the observation position. The abbreviation C indicates center part of the grains.
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FIG. 4. Comparison of the distribution of the two types of protein bodies in brown rice grains among cultivars. (A) Control; (B) LGC1; (C) Tashu-kei 1001; (D) Hyogokitanishiki; (E) Yamadanishiki; (F) Nipponbare. The control image showing the level of nonspecific fluorescence with rabbit serum albumin and anti-rabbit antibodies. The signals are as described in Fig. 3. The fluorescence due to PB-I and PB-II detected close to the cell wall in endosperm cells and appearing as yellow dots is shown in the region surrounded by the dotted line in panels B, E and F. Abbreviations: A, aleurone layer; P, pericarp; S, starchy endosperm; CW, cell wall.
FIG. 5. Comparison of the distribution of the two types of protein bodies in 70% polished rice grains among cultivars. (A) Control; (B) LGC1; (C) Tashu-kei 1001; (D) Hyogokitanishiki; (E) Yamadanishiki; (F) Nipponbare. Arrows indicate the outline of the 70% polished rice grain. Control image, signals and abbreviations are as described in Fig. 3.
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elimination by polishing. There are apparent differences in the distribution of the two types of protein bodies in 70% polished rice compared with brown rice among the cultivars used in this study. In the images for the low-glutelin rice LGC1 and Tashu-kei 1001, green signals (PB-I) are detected in the rice grains so strongly as red signals (PB-II) at the fluorescence strength (Fig. 5B, C). This suggests that a substantial amount of PB-I remained in these 70% polished rice grains despite the elimination of 30% of the endosperm tissues from the outer layer of the brown rice grain. In Hyogokitanishiki and Yamadanishiki rice suitable for sake brewing, PB-II is distributed over a wide area and PB-I over a small area (Fig. 5D, E). Figure 5D, Hyogokitanishiki, shows that yellow signals are strongly detected in the outer layer of grains and red signals at the center. This suggests that PB-I and PB-II localize close to each other in the outer layer, but most of the PB-II is localized at the center of the grain. Figure 5E shows that PB-II is distributed from the outer layer to the center of the endosperm in the 70% polished rice grains of Yamadanishiki. The distribution of PB-I and PB-II in Nipponbare rice grains is similar to that in Yamadanishiki rice grains (Fig. 5F). Thus, these observations reveal the difference in the distribution of the two types of protein bodies in endosperm tissues between low-glutelin rice and the other rice cultivars. DISCUSSION The important features of this study are as follows. The finding of the first is the difference in the distribution of the two types of protein bodies in the 70% polished grains of low-glutelin rice and the other rice cultivars. The second is the microscopic observation of the endosperm tissues of low-glutelin rice, which is probably the first investigation of storage protein accumulation using a fluorescent antibody technique. The site of accumulation of storage proteins in the 70% polished rice endosperm of the low-glutelin rice Tashu-kei 1001 was observed by light microscopy. In Fig. 2A, a large amount of storage proteins could be observed in the center of 70% polished rice grains despite the elimination of 30% of the outer layer of brown rice grains. This suggests that the distribution of storage proteins in low-glutelin rice differs essentially from that of other rice cultivars previously investigated. Therefore, the variety of rice cultivar might have a significant effect not only on the protein composition in endosperm tissues but also on the distribution of the two types of protein bodies. To visualize the distribution of the two types of protein bodies in rice grains, we observed the grains of brown rice and 70% polished rice using CLSM, with the results shown in Figs. 3 to 5. Figure 4 shows the distribution of the two types of protein bodies in brown rice, which was basically similar among all the cultivars. However, for the polished rice which is used as a raw material for brewing sake, there were marked differences in the distribution of the protein bodies in the 70% polished rice grains among some of the cultivars, as shown in Figs. 3 and 5. Previously, we determined the digestibility of seed storage proteins in sake mashes among several rice cultivars, as a characteristic of
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the raw material used for brewing sake (10). We reported that low-glutelin rice was rich in prolamin rather than glutelin (13–15) and that there is a marked difference in the digestibility of storage proteins among rice cultivars (10, 13). Our previous findings are consistent with the present results showing that there are apparent differences in the distribution of the two types of protein bodies in 70% polished rice grains among the cultivars used in this study, as shown in Fig. 5. Thus, we have concluded that the distribution of PB-I and the amount of prolamin in endosperm tissues have a significant effect on the digestion of rice seed storage proteins in sake mashes. It was previously reported by Shizukawa et al. that there was a difference in the distribution of storage proteins in rice grains between rice suitable for sake brewing and rice used for general consumption (17). In this study, the distribution of storage proteins in rice grains could be visualized and differences among the rice cultivars were revealed. PB-I was widely distributed over the endosperm tissues in low-glutelin rice. The rice grains of the other cultivars mainly consisted of PB-II, which was distributed predominantly in the center part of the endosperm tissues. These results show that there are essential differences in the protein composition and distribution in low-glutelin rice and other cultivars, though in general the majority of storage proteins are contained in the outer layer of grains. These differences are considered to be related to the endosperm intrastructure of the proteins bodies and starch granules in the endosperm or the protein composition of endosperm tissues, in particular the percentage of PB-I which includes large amounts of hydrophobic amino acids. These considerations invite further investigation. Our results propose new and important ideas regarding the distribution of the two types of protein bodies in rice grains. In the biosynthesis of the rice seed storage proteins, the expression of prolamin is later than that of glutelin in the seed development stage, and continues almost until the mature stage is reached. Thus, it has naturally been assumed that prolamin accumulates mainly in the outer layer of a rice grain. In low-glutelin rice, the LGC1 gene confers the low glutelin content phenotype by RNA silencing, but it does not affect the increasing of prolamin content (18). The mechanism underlying the protein composition of low-glutelin rice has not yet been clarified. The finding in this study that the distribution of storage proteins in the rice grains of low-glutelin rice differs from that in the grains of the other cultivars, should help clarify the biosynthesis of the storage proteins in low-glutelin rice. It should be noted that in low-glutelin rice, PB-I is distributed throughout the rice endosperm tissue despite the elimination of 30% of the outer layer of the brown rice grains. As for the sake brewing process, glutelin has been considered to enhance the taste of sake in general, because it is digestible and is distributed over a wide area of the grain. On the other hand, prolamin has not been considered to have a significant effect on the taste of sake because it is indigestible. However, we previously reported that sake brewing using low-glutelin rice for “kakemai” produced a new type of sake with a unique taste (14). We also previously determined the digestibility of seed storage proteins in sake
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mashes among several rice cultivars, and reported that prolamin which is indigestible in the human body can be digested to some extent in sake mash (10). These reports and the results in this paper suggest that prolamin in low-glutelin rice might have some effect on the taste of sake because a significant amount of prolamin remains in the center of polished rice grains. We will next examine the effect of prolamin on the taste of sake made with various types of low-glutelin rice as “kakemai”. These studies will provide further understanding of the differences in the distribution of seed storage proteins among rice cultivars and their effects on sake taste. ACKNOWLEDGMENTS We are grateful to Dr. Shuichi Iida (the National Agricultural Research Center for the Western Region) for providing the rice cultivars. We also thank to the members of the Laboratory of Genetic Engineering, Faculty of Agriculture, Kyoto Prefectural University, for supporting the present work. This research was performed at Kyoto Prefectural Institute of Agricultural Biotechnology as a fellowship of the Open Laboratory System.
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7. Kojima, H., Hasegawa, H., Magarifuchi, T., and Fukuda, H.: Observation of rice koji by confocal laser scanning microscopy. Seibutsu-kogaku, 77, 339–344 (1999). 8. Kizaki, Y., Inoue, Y., Okazaki, N., and Kobayashi, S.: Isolation and determination of protein bodies (PB-I, PB-II) in polished rice endosperm. J. Brew. Soc. Jpn., 86, 293–298 (1991). (in Japanese) 9. Kizaki, Y., Ohara, A., Henmi, A., Aramaki, I., and Kobayashi, S.: Difference in protein body components among rice cultivars used for sake brewing. J. Brew. Soc. Jpn., 88, 326– 331 (1993). (in Japanese) 10. Furukawa, S., Mizuma, T., Yanagiuchi, T., Kiyokawa, Y., and Wakai, Y.: Dissolution of protein from rice used for sake brewing in sake mash. J. Brew. Soc. Jpn., 95, 295–303 (2000). (in Japanese) 11. Iwano, K., Nakazawa, N., Ito, T., Takahashi, H., Uehara, Y., and Matsunaga, R.: The influence of protein components in raw material rice on various enzyme activities in sake-koji. J. Brew. Soc. Jpn., 96, 857–862 (2001). (in Japanese) 12. Yoshii, M. and Aramaki, I.: Microstructure of sake-koji. J. Brew. Soc. Jpn., 96, 806–813 (2001). (in Japanese) 13. Furukawa, S., Mizuma, T., Kiyokawa, Y., Yanagiuchi, T., Wakai, Y., Matsushita, K., Maeda, H., Iida, S., and Nemoto, H.: Observation of low-glutelin rice for sake brewing by scanning electron microscope. Seibutsu-kogaku, 80, 512–520 (2002). 14. Mizuma, T., Furukawa, S., Kiyokawa, Y., Wakai, Y., Yamamoto, Y., Tsutsui, N., Matsushita, K., Maeda, H., Iida, S., and Nemoto, H.: Characteristics of low-glutelin rice for sake brewing — Studies on rice for sake brewing. Seibutsu-kogaku, 80, 503–511 (2002). 15. Iida, S., Amano, E., and Nishio, T.: A rice (Oryza sativa L.) mutant having a low content of glutelin and a high content of prolamine. Theor. Appl. Genet., 87, 374–378 (1993). 16. Masumura, T. and Tanaka, K.: Biosynthesis and accumulation of rice storage proteins. J. Brew. Soc. Jpn., 88, 414–420 (1993). (in Japanese), 17. Shizukawa, Y., Oohashi, Y., Masumura, T., and Tanaka, K.: The contents and distribution of prolamin, glutelin and globulin that constitute storage protein (protein body) in brown rice on some rice cultivars for brewery or meal. Jpn. J. Crop. Sci., 71, 224–225 (2002). (in Japanese) 18. Kusaba, M., Miyahara, K., Iida, S., Fukuoka, H., Takano, T., Sassa, H., Nishimura, M., and Nishio, T.: Low glutelin content1: a dominant mutation that suppresses the glutelin multigene family via silencing in rice. Plant Cell, 15, 1455– 1467 (2003).
Distribution of Storage Proteins in Low-Glutelin Rice Seed Determined Using a Fluorescent Antibody SACHIKO FURUKAWA,1* TOMOCHIKA MIZUMA,1 YOSHIFUMI KIYOKAWA,1 TAKEHIRO MASUMURA,2,3 KUNISUKE TANAKA,2,3 AND YOSHINORI WAKAI1 Kizakura Sake Brewing Co. Ltd., 53 Kajiwara-cho, Shimomisu, Yoko-ohji, Fushimi-ku, Kyoto 612-8242, Japan,1 Laboratory of Genetic Engineering, Faculty of Agriculture, Kyoto Prefectural University, Shimogamo, Kyoto 606-8522, Japan,2 and Kyoto Prefectural Institute of Agricultural Biotechnology, Seika, Kyoto 619-0244, Japan3 Received 23 April 2003/Accepted 25 August 2003
To compare the distribution of storage proteins in low-glutelin rice seed with that in other cultivars having normal protein compositions, immunofluorescence labeling with specific antibodies was applied to visualize the distribution of storage proteins in endosperm tissues. The endosperm tissues from five cultivars were reacted with anti-prolamin and anti-glutelin antibodies, and then observed by light microscopy and confocal laser scanning microscopy (CLSM). In low-glutelin rice, using microscopic analysis, a large proportion of storage proteins was observed in the endosperm tissue of 70% polished rice. To determine the localization of two types of protein bodies in endosperm tissues, images of the distribution of the type I protein body (PB-I) and the type II protein body (PB-II) were obtained by CLSM. The CLSM images showed that, in low-glutelin rice, prolamin which accumulates in PB-I remains in the center of 70% polished rice grains despite the elimination of 30% of the outer layer of brown rice grains. However, the other cultivars mostly contained glutelin which accumulates in PB-II and is distributed throughout the endosperm tissues. This shows that low-glutelin rice differs from the other cultivars not only in the major storage protein composition but also in the distribution of storage proteins in endosperm tissues. [Key words: confocal laser scanning microscopy, protein body, fluorescent antibody technique, low-glutelin rice, sake brewing]
Observation by confocal laser scanning microscopy (CLSM) provides images with high depth and high resolution with a minimum of background noise (7). Positional relationship can be determined in the area of interest by differential staining (7). Several studies have been conducted on the behavior of rice protein bodies in the sake brewing process (8–11) and the morphology of rice grains as a raw material in sake brewing (7, 12, 13). However, to our knowledge, there has been no published study comparing the distribution of storage proteins in the endosperm tissues of rice used for brewing sake using a fluorescent antibody technique. For lowglutelin rice, of which the content of glutelin is reduced, we previously determined the effect of the composition of the rice protein on the sake brewing process, and indicated the possibility that prolamin is highly related to the dissolution of the raw material rice grain (13, 14). Therefore, we consider it would be interesting to investigate the distribution of storage proteins in the raw material rice grain and determine the effect on the sake brewing process. To visualize the distribution of rice seed storage proteins, we performed double immunofluorescence labeling with specific antibodies against prolamin and glutelin individually, and observed the results using CLSM. In this paper, we
In rice endosperm cells, seed storage proteins (proteins stored in seed) accumulate in two types of granules called protein bodies. It has been shown that the type I protein body (PB-I) stores prolamin as an alcohol-soluble protein, whereas the type II protein body (PB-II) mainly contains glutelin as an alkaline (and/or acid)-soluble protein (1, 2). PB-I cannot be digested by the human digestive system, whereas PB-II is digestible (3–5). Generally, rice protein bodies (japonica rice) consist of 20–30% PB-I and 70–80% PB-II. Rice is a staple food in Japan, and often used as a raw material for brewing Japanese sake. The function of rice grains in the sake brewing process is as a growth medium for yeast such as “kakemai” or koji as “rice-koji”. Free amino acids and peptides liberated from rice seed proteins are known to play an important role in enhancing the taste of sake (6). Rice grains containing small amounts of protein are suitable for brewing sake because surplus amino acids spoil the taste. Therefore, analytical information on rice seed proteins is indispensable to selecting rice cultivars as raw materials in sake brewing. * Corresponding author. e-mail: [email protected] phone: +81-(0)75-611-4101 fax: +81-(0)75-622-3510 467
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TABLE 1. The list of rice cultivars used in this study Crude protein contents (g/100 g) Protein composition of 70% polished rice (%) Brown rice 70% polished rice PB-I PB-II Others LGC1 * 7.2 5.1 41.4 36.7 21.9 Tashu-kei 1001 * 7.6 5.0 39.4 38.5 22.1 Hyogokitanishiki * 8.3 5.2 20.4 59.1 20.5 Yamadanishiki Hyogo 7.1 4.5 22.5 58.0 19.5 Nipponbare Shiga 7.9 5.2 22.3 58.7 19.0 * The National Agricultural Research Center for the Western Region. Protein composition was analyzed according to Kizaki et al. (8). PB-I represents the type I protein body and PB-II represents the type II protein body. Rice cultivars
Locality
report the different distributions of storage proteins in the rice grains of low-glutelin rice and other cultivars used for brewing sake.
labeled samples. The processing images for the localization of PB-I (prolamin) and PB-II (glutelin) in endosperm cells were obtained separately and merged.
MATERIALS AND METHODS
RESULTS
Materials Table 1 shows the rice (Oryza sativa L.) cultivars used in this study, which were harvested in 2000. Three cultivars listed in Table 1, i.e., LGC1, Tashu-kei 1001 and Hyogokitanishiki, were from the National Agricultural Research Center for the Western Region. LGC1, which contains small amounts of glutelin and large amounts of prolamin, is a low-glutelin rice used in the diet of patients with kidney disease (15). Tashu-kei 1001 was cultivated as a hybrid of LGC1 and Hyogokitanishiki to be used for sake brewing, and genetically has the protein composition of LGC1 (13, 14). To obtain polished rice for sake brewing, brown rice was polished to 70% of its original weight using a pearling mill (the graintesting mill; Satake, Higashi-Hiroshima). SDS–PAGE of brown rice and 70% polished rice proteins The rice seed storage proteins were extracted from the grains of brown rice and 70% polished rice (polished to 70% of its original weight) according to the methods of Kizaki et al. (8), and separated by SDS–PAGE using 15% acrylamide gels. The gels were stained with Coomassie Brilliant Blue R-250 (CBB R-250), and the protein composition was analyzed using a densitometer (Densitograph AE-6920M; ATTO, Tokyo). Preparation of cross sections A single grain of brown rice or 70% rice was cross-sectioned with a laser blade. The cross section was attached to a glass slide for observation under a fluorescence microscope. Then, thin cross sections of 200 mm were cut with a sliding microtome (HM400R; Microm, Walldolf, Germany). Observations by CLSM Immunofluorescence labeling using specific antibodies against prolamin and glutelin was conducted as follows. Cross sections of rice grains were fixed in 4.0% (w/v) paraformaldehyde and 0.5% (v/v) glutaraldehyde in phosphate buffer (PBS, pH 7.2), then blocked in 1.0% (w/v) BSA in TBST (0.05% [w/v] Tween 20 and 150 mM NaCl in 10 mM Tris–HCl [pH 8.0]) at room temperature for 1 h. Then, the fixed specimens were washed twice with TBST for 10 min at room temperature. After fixation and washing, tissue sections were reacted with a specific antibody from rabbit against the 13 kDa prolamin and from mouse against the 23 kDa glutelin for 1 h at 37°C. Reacted tissue sections were washed three times with TBST for 10 min, then, the secondary antibody was subsequently reacted with a FluoroLink™ Cy™2 (FITC)-labeled goat anti-rabbit IgG (Amersham Biosciences, Tokyo) and a FluoroLink™ Cy™3 (Rhodamine)-labeled goat anti-mouse IgG (Amersham Biosciences) for 1 h at 37°C. After washing with TBST as described above, tissue sections were observed using CLSM. A CLSM system (MRC-1024; Bio-Rad Laboratories, Hercules, CA, USA) was used for observing double immunofluorescence
Comparison of protein composition among rice cultivars Table 1 shows the crude protein contents of the brown rice and 70% polished rice used in this study. The crude protein content of Yamadanishiki 70% polished rice is 4.5 (g/100 g) which is lower than that of the other cultivars for which the crude protein content is approximately 5.0 (g/100 g). The storage proteins of brown rice and 70% polished rice were separated by SDS–PAGE. Figure 1 shows the SDS– PAGE protein band patterns of brown rice and 70% polished rice (cv. Hyogokitanishiki, LGC1 and Tashu-kei 1001). A comparison of these three cultivars showed that the protein band patterns of the brown rice and 70% polished rice exhibited distinct features. More precisely, in LGC1 and Tashu-kei 1001, the prolamin (10 kDa, 13 kDa, and 16 kDa polypeptides) content increased while the glutelin (22–23 kDa and 37–39 kDa polypeptides) content decreased compared to in Hyogokitanishiki. The crude protein content of
FIG. 1. SDS–PAGE protein band patterns of brown rice and 70% polished rice grains. Lane 1, Hyogokitanishiki; 2, LGC1; 3, Tashu-kei 1001; 4, Hyogokitanishiki; 5, LGC1; 6, Tashu-kei 1001. Lanes 1 to 3, Brown rice; lanes 4 to 6, 70% polished rice.
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Hyogokitanishiki brown rice was 8.3 (g/100 g), which is higher than those of LGC1 and Tashu-kei 1001; however, there was little difference among these cultivars in the total protein content of 70% polished rice, as shown in Table 1. The protein band patterns of Yamadanishiki and Nipponbare were similar to that of Hyogokitanishiki (data not shown). The protein compositions of the 70% polished rice were determined from the SDS–PAGE protein band patterns of these five cultivars, and are shown in Table 1. In LGC1 and Tashu-kei 1001, the protein composition consisted of higher level of PB-I and lower level of PB-II in comparison with the others. Thus, low-glutelin rice differed from Hyogokitanishiki and Yamadanishiki rice suitable for sake brewing and Nipponbare rice used for general consumption in the major protein composition. Distribution of storage proteins in low-glutelin rice endosperm To examine the distribution of storage proteins in low-glutelin rice, 70% polished rice was observed by light microscopy (BH-2; Olympus, Tokyo). Figure 2 shows the micrographs of a cross section of the 70% polished rice endosperm of Tashu-kei 1001 (Fig. 2A) and Hyogokitanishiki (Fig. 2B). In Fig. 2A, it is possible to clearly distinguish the area in which the storage proteins are stained blue with CBB (indicated by arrows as PB), and the starch granules observed as white. A large proportion of storage proteins could be observed in the outer layer of the 70% polished rice grain (the upper side of Fig. 2A). However, in Fig. 2B, the storage proteins stained with CBB could be observed to be spread thinly over the entire. Generally, PB-II is mostly distributed throughout the endosperm tissues, whereas PB-I is mostly confined to the outer layer of the grain (16, 17). Therefore, in the sake brewing process, polishing is useful for eliminating storage proteins from the outer layer of grains as is well known. That is, 70% polished rice consists almost completely of the starchy endosperm tissues without the aleurone or subaleurone layer because of the elimination of 30% of the outer layer of brown rice grains. However, in the lowglutelin rice Tashu-kei 1001, a large amount of storage proteins could be observed in the 70% polished rice as shown in Fig. 2A. This suggests that the distribution of storage proteins in the low-glutelin rice endosperm differs fundamentally from that of the other rice cultivars as shown in Fig. 2B. Distribution of two types of protein bodies in rice endosperm using CLSM To visualize the distribution of rice seed storage proteins, we used CLSM. Figure 3 shows CLSM cross-sectional images of 70% polished rice grains (cv. LGC1) treated with double fluorescence labeling using the anti-prolamin and anti-glutelin antibodies. Figure 3A–C is images of the storage proteins from the same rice grains. The distribution of prolamin (green) and glutelin (red) is displayed separately in Fig. 3A and 3B, but the two images are merged in Fig. 3C, thus the overlapping regions appear yellow. To determine the extent of nonspecific fluorescence generated by the antibody reaction, the fluorescence produced using only the primary or secondary antibody was detected. Figures 4A and 5A show sample results which indicate that the level of nonspecific fluorescence generated
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by the antibody reaction was very low and did not affect the detection of fluorescence with respect to PB-I and PB-II. The distribution of storage proteins in the endosperm tissues of steamed rice and koji was previously reported by Kojima et al. (7). However, in this study, Fig. 3 shows the distribution of prolamin and glutelin in a rice grain. The distribution of the two types of protein bodies in brown rice Figure 4 shows the distributions of the two types of protein bodies in brown rice. Half of the grains were compared between the five cultivars shown in Table 1 using the CLSM merged images. Figure 4A, as the control image, reveals that the level of nonspecific fluorescence generated by the secondary antibody reaction to detect prolamin is very weak. In the other control images, the level of nonspecific fluorescence generated by the secondary antibody reaction is similar to that in Fig. 4A (data not shown). This means that the nonspecific fluorescence slightly affects the fluorescence generated due to the reaction of the antiprolamin and anti-glutelin antibodies under the experimental conditions described above. The merged images of the double fluorescence labeling with the anti-prolamin and anti-glutelin antibodies are as follows: LGC1 (Fig. 4B), Tashu-kei 1001 (Fig. 4C), Hyogokitanishiki (Fig. 4D), Yamadanishiki (Fig. 4E), Nipponbare (Fig. 4F). The storage proteins of brown rice, shown in yellow, are distributed in the outer layer of grains in all rice cultivars in Fig. 4. Notably, Fig. 4B, 4E and 4F clarify that the storage proteins (PB-I and PB-II) are located close to the cell walls in the endosperm cells, shown as yellow dots (inside the region surrounded by the dotted line). These results indicate that the storage proteins are mostly localized close to the cell walls in endosperm tissues, which surround starch granules (13, 16). In addition, Fig. 4 shows that the brown rice grains of the five cultivars appear to differ slightly in the distribution of storage proteins. That is, the proteins are mostly distributed on the outer layer of the grains. The distribution of the two types of protein bodies in 70% polished rice Because high polished rice is used as a raw material for sake brewing, investigation of the distribution of the two types of protein bodies in 70% polished rice is more important than that in brown rice. Figure 5 shows the distribution of the two types of protein bodies in 70% polished rice of all the cultivars used in this study. Figure 5B to 5F represents the same cultivars as represented in Fig. 4B to 4F. Figure 5A is the control image similar to that in Fig. 4A, and reveals that the nonspecific fluorescence generated in double staining has a little effect on the fluorescence obtained with anti-prolamin (PB-I) and anti-glutelin (PB-II) in 70% polished rice. In 70% polished rice, storage proteins are distributed throughout the endosperm tissues of every cultivars as shown in Fig. 5. It is well known that starch granules generally occupy most of the rice endosperm. However, these results show that the storage proteins are also important as major components even in the 70% polished rice. In this study, based on the size of a rice grain, the 30% eliminated by polishing represents about 500 mm from the outer layer of the brown rice grain. Therefore, most of the area shown in Fig. 4 does not exist in Fig. 5 because of
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FIG. 2. Light microscopic images of 70% polished rice endosperm of low-glutelin rice. (A) Tashu-kei 1001; (B) Hyogokitanishiki. Storage proteins are stained blue with CBB (arrows indicate PB). The starch granules appear as white spheres. The images show the outer layer of a 70% polished rice grain (upper), and close to the center of a 70% polished rice grain (lower). PB represents protein bodies. The schematic shown below the images demonstrates the observation position (side of cross section of 70% polished rice grains).
FIG. 3. Double fluorescence labeling of brown rice and 70% polished rice grains using CLSM. (A) Anti-prolamin antibody to determine distribution of PB-I; (B) anti-glutelin antibody to determine distribution of PB-II; (C) merged image. The distribution of PB-I (prolamin) and PB-II (glutelin) in the rice grains (cultivar, LGC1) was determined by fluorescence labeling. In Fig. 2A, the green signal is due to a reaction with a FluoroLink™ Cy™2 (FITC)-labeled goat anti-rabbit IgG and in Fig. 2B, the red signal to a reaction with a FluoroLink ™ Cy™3 (Rhodamine)-labeled goat anti-mouse IgG, which indicate the distribution of PB-I and PB-II, respectively. Yellow indicates the overlapping regions. Arrows indicate the outline of the 70% polished rice grain. The schematic shown below the images demonstrates the observation position. The abbreviation C indicates center part of the grains.
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FIG. 4. Comparison of the distribution of the two types of protein bodies in brown rice grains among cultivars. (A) Control; (B) LGC1; (C) Tashu-kei 1001; (D) Hyogokitanishiki; (E) Yamadanishiki; (F) Nipponbare. The control image showing the level of nonspecific fluorescence with rabbit serum albumin and anti-rabbit antibodies. The signals are as described in Fig. 3. The fluorescence due to PB-I and PB-II detected close to the cell wall in endosperm cells and appearing as yellow dots is shown in the region surrounded by the dotted line in panels B, E and F. Abbreviations: A, aleurone layer; P, pericarp; S, starchy endosperm; CW, cell wall.
FIG. 5. Comparison of the distribution of the two types of protein bodies in 70% polished rice grains among cultivars. (A) Control; (B) LGC1; (C) Tashu-kei 1001; (D) Hyogokitanishiki; (E) Yamadanishiki; (F) Nipponbare. Arrows indicate the outline of the 70% polished rice grain. Control image, signals and abbreviations are as described in Fig. 3.
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elimination by polishing. There are apparent differences in the distribution of the two types of protein bodies in 70% polished rice compared with brown rice among the cultivars used in this study. In the images for the low-glutelin rice LGC1 and Tashu-kei 1001, green signals (PB-I) are detected in the rice grains so strongly as red signals (PB-II) at the fluorescence strength (Fig. 5B, C). This suggests that a substantial amount of PB-I remained in these 70% polished rice grains despite the elimination of 30% of the endosperm tissues from the outer layer of the brown rice grain. In Hyogokitanishiki and Yamadanishiki rice suitable for sake brewing, PB-II is distributed over a wide area and PB-I over a small area (Fig. 5D, E). Figure 5D, Hyogokitanishiki, shows that yellow signals are strongly detected in the outer layer of grains and red signals at the center. This suggests that PB-I and PB-II localize close to each other in the outer layer, but most of the PB-II is localized at the center of the grain. Figure 5E shows that PB-II is distributed from the outer layer to the center of the endosperm in the 70% polished rice grains of Yamadanishiki. The distribution of PB-I and PB-II in Nipponbare rice grains is similar to that in Yamadanishiki rice grains (Fig. 5F). Thus, these observations reveal the difference in the distribution of the two types of protein bodies in endosperm tissues between low-glutelin rice and the other rice cultivars. DISCUSSION The important features of this study are as follows. The finding of the first is the difference in the distribution of the two types of protein bodies in the 70% polished grains of low-glutelin rice and the other rice cultivars. The second is the microscopic observation of the endosperm tissues of low-glutelin rice, which is probably the first investigation of storage protein accumulation using a fluorescent antibody technique. The site of accumulation of storage proteins in the 70% polished rice endosperm of the low-glutelin rice Tashu-kei 1001 was observed by light microscopy. In Fig. 2A, a large amount of storage proteins could be observed in the center of 70% polished rice grains despite the elimination of 30% of the outer layer of brown rice grains. This suggests that the distribution of storage proteins in low-glutelin rice differs essentially from that of other rice cultivars previously investigated. Therefore, the variety of rice cultivar might have a significant effect not only on the protein composition in endosperm tissues but also on the distribution of the two types of protein bodies. To visualize the distribution of the two types of protein bodies in rice grains, we observed the grains of brown rice and 70% polished rice using CLSM, with the results shown in Figs. 3 to 5. Figure 4 shows the distribution of the two types of protein bodies in brown rice, which was basically similar among all the cultivars. However, for the polished rice which is used as a raw material for brewing sake, there were marked differences in the distribution of the protein bodies in the 70% polished rice grains among some of the cultivars, as shown in Figs. 3 and 5. Previously, we determined the digestibility of seed storage proteins in sake mashes among several rice cultivars, as a characteristic of
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the raw material used for brewing sake (10). We reported that low-glutelin rice was rich in prolamin rather than glutelin (13–15) and that there is a marked difference in the digestibility of storage proteins among rice cultivars (10, 13). Our previous findings are consistent with the present results showing that there are apparent differences in the distribution of the two types of protein bodies in 70% polished rice grains among the cultivars used in this study, as shown in Fig. 5. Thus, we have concluded that the distribution of PB-I and the amount of prolamin in endosperm tissues have a significant effect on the digestion of rice seed storage proteins in sake mashes. It was previously reported by Shizukawa et al. that there was a difference in the distribution of storage proteins in rice grains between rice suitable for sake brewing and rice used for general consumption (17). In this study, the distribution of storage proteins in rice grains could be visualized and differences among the rice cultivars were revealed. PB-I was widely distributed over the endosperm tissues in low-glutelin rice. The rice grains of the other cultivars mainly consisted of PB-II, which was distributed predominantly in the center part of the endosperm tissues. These results show that there are essential differences in the protein composition and distribution in low-glutelin rice and other cultivars, though in general the majority of storage proteins are contained in the outer layer of grains. These differences are considered to be related to the endosperm intrastructure of the proteins bodies and starch granules in the endosperm or the protein composition of endosperm tissues, in particular the percentage of PB-I which includes large amounts of hydrophobic amino acids. These considerations invite further investigation. Our results propose new and important ideas regarding the distribution of the two types of protein bodies in rice grains. In the biosynthesis of the rice seed storage proteins, the expression of prolamin is later than that of glutelin in the seed development stage, and continues almost until the mature stage is reached. Thus, it has naturally been assumed that prolamin accumulates mainly in the outer layer of a rice grain. In low-glutelin rice, the LGC1 gene confers the low glutelin content phenotype by RNA silencing, but it does not affect the increasing of prolamin content (18). The mechanism underlying the protein composition of low-glutelin rice has not yet been clarified. The finding in this study that the distribution of storage proteins in the rice grains of low-glutelin rice differs from that in the grains of the other cultivars, should help clarify the biosynthesis of the storage proteins in low-glutelin rice. It should be noted that in low-glutelin rice, PB-I is distributed throughout the rice endosperm tissue despite the elimination of 30% of the outer layer of the brown rice grains. As for the sake brewing process, glutelin has been considered to enhance the taste of sake in general, because it is digestible and is distributed over a wide area of the grain. On the other hand, prolamin has not been considered to have a significant effect on the taste of sake because it is indigestible. However, we previously reported that sake brewing using low-glutelin rice for “kakemai” produced a new type of sake with a unique taste (14). We also previously determined the digestibility of seed storage proteins in sake
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mashes among several rice cultivars, and reported that prolamin which is indigestible in the human body can be digested to some extent in sake mash (10). These reports and the results in this paper suggest that prolamin in low-glutelin rice might have some effect on the taste of sake because a significant amount of prolamin remains in the center of polished rice grains. We will next examine the effect of prolamin on the taste of sake made with various types of low-glutelin rice as “kakemai”. These studies will provide further understanding of the differences in the distribution of seed storage proteins among rice cultivars and their effects on sake taste. ACKNOWLEDGMENTS We are grateful to Dr. Shuichi Iida (the National Agricultural Research Center for the Western Region) for providing the rice cultivars. We also thank to the members of the Laboratory of Genetic Engineering, Faculty of Agriculture, Kyoto Prefectural University, for supporting the present work. This research was performed at Kyoto Prefectural Institute of Agricultural Biotechnology as a fellowship of the Open Laboratory System.
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