Localization of Pullulanase- and α-glucosidase-like Immuno-reactivity in Non-glutinous Rice Grains during Cooking

Abstract

The pullulanase and α-glucosidase in brown rice is predominately found in the endosperm of rice grains of Koshihikari and Nipponbare cultivars. However, the exact localization of these enzymes in the endosperm layers is not clear. To investigate enzyme localization in non-glutinous brown rice, immuno-fluorescence detection of these enzymes was carried out using frozen sections of rice grains (Koshihikari, Nipponbare and Milkyqueen cultivars). While pullulanase-like immuno-reactivity was dominantly detected in amyloplasts, α-glucosidase-like immuno-reactivity was mainly detected along endosperm cell walls among the three non-glutinous cultivars. Immuno-reactivity of both enzymes overlapped in the aleurone layer and embryo among the three cultivars. During rice cooking, pullulanase-like immuno-reactivity partly overlapped with α-glucosidase-like immuno-reactivity along endosperm cell walls among the three cultivars. These results indicate that each enzyme has a unique localization, and these unique localizations are common among the three non-glutinous cultivars.

Introduction

Rice (Oryza sativa) is a staple food in Asian countries. Extensive studies on the preferable tastes of cooked rice revealed that physical properties, such as stickiness and hardness (Iwata et al., 2001; Kainuma, 1992; Maruyama, 1991; Okabe, 1979), and chemical components, such as sugars and amino acids, contribute to the taste of cooked rice (Ikeda, 2001; Matsuzaki et al., 1992; Tajima et al., 1992; Uyen Tran et al., 2004). The amounts of sugars (reducing sugars and oligosaccharides) and amino acids are correlated with the activities of starch-degrading enzymes and proteases, which are activated during cooking (Kasai et al., 2000; Kishio and Aoyagi, 2014; Maruyama, 1991; Maruyama, 2002). Kasai et al. revealed that reducing sugars and glucose showed the largest increase at a temperature range of 40–60 °C during cooking in both rice grains and cooking water. Mabashi et al. analyzed the hydrolytic activities of endogenous starch-degrading enzymes for soluble starch among various cultivars (Koshihikari, Nipponbare, Habutaemochi, Yumetoiro and Milkyqueen), and revealed that 60 °C was the common key temperature for the production of glucose by endogenous enzymes in various cultivars (Mabashi et al., 2009). Kishio and Aoyagi revealed that endosperm α-glucosidase activity was higher than α-amylase and-β-amylase like activity among various cultivars (Koshihikari, Hinohikari, Akitakomachi, Hitomebore and Kinuhikari). However, localization of the corresponding starch-degrading enzymes in rice grains remains unclear at a temperature range of 40–60 °C. Investigation of the behavior of rice starch-degrading enzymes during rice cooking is thus worth pursuing.

The α-glucosidases (EC 3.2.1.20) are enzymes that produce glucose from the non-reducing ends of α-1,4-glucans in starch, and this activity has been reported to correlate with the physical and chemical properties of rice (Iwata et al., 2002). Isoamylases and pullulanase are enzymes that hydrolyze α-1,6-glucosidic linkages of amylopectin. In Koshihikari, Nipponbare and Habutaemochi cultivars, we previously revealed that α-glucosidase, pullulanase and isoamylases were predominantly localized at the inner endosperm layers in brown rice grains (Tsuyukubo et al., 2013; Tsuyukubo et al., 2010; Tsuyukubo et al., 2012). We, however, omitted Milkyqueen — which has lower amylose content than Koshihikari — from the previous analysis due to its fragility on milling. We also excluded polished rice grains from the cooking process, as these rice grains were too soft to fractionate by milling. Thus, the localization of enzymes in Milkyqueen and cooked rice remains unclear. Recently, Saito et al. reported the utility of Kawamoto's thin frozen film method (Kawamoto, 2003) in determining the immuno-localization of the 13-kDa prolamin in whole rice grains (Saito et al., 2008). Applying this method to three non-glutinous brown and cooked rice (Koshihikari, Nipponbare and Milkyqueen), we here report the localization of pullulanase- and α-glucosidase-like immuno-reactivity using an immuno-fluorescence microscopy technique.

Materials and Methods

Materials    Reagents and chemicals were purchased from the following commercial sources: protein markers from New England Biolabs (Ipswich, USA), bis-acrylamide and 35% EmpigenBB from Sigma Aldrich (St. Louis, USA), protein assay kit from Bio-Rad (Hercules, USA), and acrylamide, horseradish peroxidase-labeled anti-rabbit IgG antibody and chemi-luminescent reagent from GE Healthcare (Buckinghamshire, England). StartingBlock blocking solution in PBS, Alexa 488-labeled anti-mouse IgG antibody, Alexa 594-labeled anti-rabbit IgG antibody and magnetic Protein-G beads were from Thermo Fisher Scientific (Tokyo, Japan). Other reagents were purchased from Fujifilm Wako Pure Chemicals (Osaka, Japan), unless otherwise noted.

Rice samples    The rice samples in this study were Oryza sativa L. cv Koshihikari (grown at Ibaraki in 2013), Nipponbare (grown at Shiga in 2012), and Milkyqueen (grown at Ibaraki in 2012), which were stored at 4 °C until use. The brown rice was polished at 90% with a VP-32T (Yamamoto Test Whitening Machine, Yamagata, Japan). The polished rice was soaked in 1.5 times cold water for 16 h at 8 °C, then cooked with an electric rice cooker, Tacook (Tiger, Tokyo, Japan). When the temperature reached 40 °C or 60 °C, samples of rice grains and cooking water were removed, and the rice grains were separated from the cooking water.

Polyclonal antibodies and monoclonal antibody production for pullulanase    The pullulanase was purified from rice powder extract with a Resource Q 5/5 anion exchange column as previously described (Tsuyukubo et al., 2012). The polyclonal antibodies against pullulanase (82-1) and the peptide-fragment (QDVIPRPSPDSFLA) of α-glucosidase (52-2) were as previously reported (Tsuyukubo et al., 2010).

The anti-pullulanase monoclonal antibody 2G11bC was obtained by immunization of C57/Black6 mice with the purified pullulanase. All experimental protocols were approved by the Committee for Animal Experimentation of the National Food Research Institute (permission number H24-004) and were in accordance with the laws of the Japanese government. Hybridomas were formed by fusion with P3U1 myeloma cells, and tissue culture, cloning, and screening procedures were essentially as described (Kohler and Milstein, 1975). To demonstrate the specificity of 2G11bC, immunoprecipitation was carried out in the presence of EmpigenBB at 0.35% from rice powder extract. After washing extensively, the bound fraction on protein-G beads was eluted by boiling in SDS-PAGE sample buffer, analyzed by SDS-PAGE, and then transferred to a PVDF membrane (Millipore, USA). The membrane was then blocked with StartingBlock blocking solution and probed with 82-1. The bound antibodies were detected with peroxidase-conjugated goat anti–rabbit IgG antibodies, and protein bands were visualized by an ECL plus detection kit (GE Healthcare).

Immuno-fluorescence microscopy    Rice grains were fixed for 3 d in 4% paraformaldehyde/phosphate buffered saline (PBS) under vacuum at 4 °C. The tissue was then embedded in SCEM (Leica Microsystems, Tokyo, Japan). Cryosections (5 µm) were mounted onto 2C(9) cryofilms (Leica Microsystems) and frozen at −30 °C until further use. Standard immunohistochemical procedures were used. Briefly, cryosections were blocked in StartingBlock (PBS) blocking solution in PBS for 1 day at 4 °C, and then incubated in primary antibody (α-glucosidase polyclonal; 1:2 000 dilution, pullulanase monoclonal 2G11bC; 2 µg/mL) in Can Get Signal Solution 1 (Toyobo, Osaka, Japan). The sections were incubated in secondary antibodies (anti-mouse-Alexa 488; 1:1 000 dilution, anti-rabbit-Alexa 594; 1:1 000) for 1 h at room temperature. After incubation, sections were washed 3 times for 5 min and coverslipped with a 3:1 mixture of SCMM-G1 (Leica Microsystems) and Fluoromount (Biomeda, Foster City, USA). Control slides were treated either without the primary antibody or with normal rabbit serum replacing the primary antiserum. Immuno-fluorescence specificity of 2G11bC was confirmed in comparison with the 82-1 polyclonal antibody. The fluorescence images were taken with a BX-810 microscope (Keyence, Tokyo, Japan), and the images were processed with Image J 1.44p with Bio-format-plugin and Photoshop CS 3 (Adobe systems, USA).

Results and Discussion

Pullulanase-like immuno-reactivity was predominant around starch granules in amyloplasts of non-glutinous brown rice    Making a longitudinal section of the rice grains as shown in Figure 1A, we analyzed pullulanase localization in the brown rice grains of Koshihikari, Nipponbare and Milkyqueen with an immuno-fluorescence microscopy technique. The longitudinal sections of brown rice grains were immunolabeled as described in the Materials and Methods.

Fig. 1.

Schematic diagram of the longitudinal sections of brown rice grains. A shows schematic diagram of the longitudinal sections of brown rice grains after the fixation and mounting as described in Materials and Methods. B shows schematic diagram of corresponding dorsal and central region the endosperm layers.

Figure 2 shows images of the pullulanase-like immuno-reactivity in whole grains, and the dorsal and ventral regions of grains. In whole grains, prominent fluorescence was observed in the endosperm layers and embryo — especially at the plumule and coleoptile. In magnified images, pullulanase-like immuno-reactivity was dominantly detected in amyloplasts and partly detected along the endosperm cell walls of Koshihikari (arrows in Fig. 2B and 2C) and Nipponbare (arrows in Fig. 2E and 2F). Although lower intensity was observed in Milkyqueen (arrows in Fig. 2H and 2I), the immuno-reactivity was dominantly detected in amyloplasts and partly along endosperm cell walls. Nakamura et al. reported that pullulanase not only hydrolyzes amylopectin in the endosperm starch during germination, but also plays essential roles in amylopectin biosynthesis (Nakamura et al., 1996). The dominant localization around starch granules might reflect its pivotal roles in germination and/or biosynthesis.

Fig. 2.

Pullulanase-like immuno-reactivity was detected in endosperm and aleurone layers of non-glutinous rice grains. The longitudinal sections of brown rice grains were immunolabeled with the monoclonal anti-pullulanase mouse IgG. Anti-mouse IgG conjugated with Alexa480 was used as a secondary antibody. The images for Koshihikari were shown at A, B, and C—those for Nipponbare at D, E, and F—those for Milkyqueen at G, H and I. Pullulanase-like immuno-reactivity was detected in endosperm and aleurone layers in Koshihikari (A), Nipponbare (D), and Milkyqueen (G). Scale bars are 0.5 mm in A, D and G. In magnified images, pullulanase-like immuno-reactivity was detected in the dorsal (arrows in B, E, and H) and central (arrows in C, F, and I) endosperm layers. Scale bars are 40 µm.

α-Glucosidase-like immuno-reactivity was predominant along endosperm cell walls in endosperm layers of non-glutinous brown rices    We analyzed α-glucosidase localization in the brown rice grains of Koshihikari, Nipponbare and Milkyqueen as described above. Figure 3 shows images of α-glucosidase-like immuno-reactivity in whole grains, and in the dorsal and central regions of grains. In whole grains, eminent fluorescence was observed in the aleurone layer and embryo — especially at the plumule, coleoptile, and epiblast. In magnified images, α-glucosidase-like immuno-reactivity was mainly detected along endosperm cell walls and outside of amyloplasts in the endosperm cell layers of Koshihikari (arrows in Fig. 3B and 3C), Nipponbare (arrows in Fig. 3E and 3F) and Milkyqueen (arrows in Fig. 3H and 3I). In contrast to the localization of pullulanase-like immuno-reactivity around starch granules in Figure 2, α-glucosidase-like immuno-reactivity was mainly localized outside of amyloplasts and along endosperm cell walls. In the embryo, pullulanase-like immuno-reactivity was restricted to the plumule and coleoptile — α-glucosidase-like immuno-reactivity was localized at the plumule, coleoptile, and epiblast. In rice germination, first α-, β-amylase, pullulanase and isoamylase degrade starch into oligosaccharides, then α-glucosidase produces glucose from oligosaccharides. In this starch degradation sequence, differences in the localization of pullulanase and α-glucosidase in the endosperm layers and embryo may contribute to consistent glucose production during germination and rice cooking.

Fig. 3.

α-Glucosidase-like immuno-reactivity was detected at endosperm and aleurone layers of non-glutinous rice grains. The longitudinal sections of brown rice grains were immunolabeled with the polyclonal anti-α-glucosidase rabbit IgG. Anti-rabbit IgG conjugated with Alexa594 was used as a secondary antibody. The images for Koshihikari were shown at A, B, and C—those for Nipponbare at D, E, and F—those for Milkyqueen at G, H and I. α-Glucosidase-like immuno-reactivity was detected in endosperm and aleurone layers in Koshihikari (A), Nipponbare (D), and Milkyqueen (G). Scale bars are 0.5 mm in A, D and G. In magnified images, α-glucosidase-like immuno-reactivity was detected along endosperm cell walls in the dorsal (arrows in B, E, and H) and central (arrows in C, F, and I) endosperm layers. Scale bars are 40 µm.

Immuno-reactivity of both enzymes was predominant at amyloplast stromal cells in the aleurone layer    We analyzed the localization of pullulanase and α-glucosidase in the dorsal and ventral aleurone layer of the brown rice grains of Koshihikari, Nipponbare and Milkyqueen using double-labeled immuno-fluorescence detection. Figure 4 shows images of pullulanase-like (green) and α-glucosidase-like (red) immuno-reactivity in the dorsal and ventral aleurone layer of Koshihikari, Nipponbare and Milkyqueen grains. We detected these enzymes co-localized at stromal cells of the aleurone layer in the dorsal (yellow in Fig. 4A, 4C, and 4E) and ventral regions of grains (yellow in Fig. 4B, 4D, and 4F). The pullulanase- and α-glucosidase-like immuno-reactivities were predominantly detected at stromal cells in the aleurone layer of the three non-glutinous cultivars. Interestingly, the width of the aleurone layer of Milkyqueen was greater than those of Koshihikari and Nipponbare. The number of aleurone cell layers of Milkyqueen in the dorsal region was 5 to 7, while that for Koshihikari and Nipponbare was 3 to 5 and 2 to 3, respectively. The data for Koshihikari were consistent with that previously reported by Hoshikawa (Hoshikawa, 1967a, Hoshikawa, 1967b). The number of aleurone cell layers of Milkyqueen was greater than that of javanica varieties (Hoshikawa, 1968), whose amylose content was greater than that of Koshihikari (Inouchi et al., 2005).

Fig. 4.

α-Glucosidase- and pullulanase-like immuno-reactivity was overlapped at aleurone layers of non-glutinous rice. The longitudinal sections of brown rice grains were immunolabeled with the polyclonal anti-α-glucosidase rabbit IgG and monoclonal anti-pullulanase mouse IgG. Anti-mouse IgG conjugated with Alexa480 and anti-rabbit IgG conjugated with Alexa594 were used as secondary antibodies. The images for Koshihikari were shown at A and B — those of Nipponbare at C and D — those of Milkyqueen at E and F. α-Glucosidase- and pullulanase-like immuno-reactivity was detected around starch granules in ventral and dorsal aleurone layers in Koshihikari (A and B), Nipponbare (C and D), and Milkyqueen (E and F). Scale bars are 200 µm.

Pullulanase-like immuno-reactivity was detected along endosperm cell walls and around starch granules in amyloplasts during cooking of non-glutinous polished rices    Making a longitudinal section of the polished rice grains as shown in Figure 1B, we analyzed the pullulanase distribution in polished rice grains during the cooking of Koshihikari, Nipponbare and Milkyqueen.

Figure 5 shows images of the pullulanase-like immuno-reactivity in Koshihikari whole grains, and dorsal, central and ventral regions of grains after the soaking step, when the temperature reached 40 °C, and when the temperature reached 60 °C. In magnified images, pullulanase-like immuno-reactivity was detected along endosperm cell walls and around starch granules at the dorsal (arrows in Fig. 5B, 5F, and 5J), central (arrows in Fig. 5C, 5G, and 5K), and ventral regions (arrows in Fig. 5D, 5H, and 5L) of the endosperm layers of Koshihikari. In comparison to Figure 2B and 2C, the immuno-reactivity around starch granules was decreased and the reactivity along endosperm cell walls was more prominent at the central region of the grains (Fig. 5C, 5G, and 5K). At the ventral region of the grains, immuno-reactivity around starch granules was detected even when the temperature reached at 60 °C (Fig. 5D, 5H, and 5L).

Fig. 5.

Pullulanase-like immuno-reactivity was detected along endosperm cell walls and around starch granules during the cooking process in Koshihikari cultivars. The longitudinal sections of polished rice grains were immunolabeled with monoclonal anti-pullulanase mouse IgG. Anti-mouse IgG conjugated with Alexa488 were used as a secondary antibody. The images for Koshihikari after the soaking step were shown at A (whole grains), B (dorsal region), C (central region), and D (ventral region) — those images when the temperature reached 40 °C shown at E, F, G, and H — those images when the temperature reached 60 °C shown at I, J, K, and L. Scale bars are 0.5 mm in A, E and I. In magnified images, pullulanase-like immuno-reactivity was partly overlapped along endosperm cell walls in the dorsal (arrows in B, F, and I), central (arrows in C, G, and K) and ventral (arrows in D, H, and L) endosperm layers. Arrows indicate representatives of positively stained areas. Scale bars are 40 µm.

Figure 6 shows images of pullulanase-like immuno-reactivity in Nipponbare whole grains, and dorsal, central and ventral regions of grains after the soaking step (Fig. 6A, 6B, 6C, and 6D), when the temperature reached 40 °C (Fig. 6E, 6F, 6G, and 6H), and when the temperature reached 60 °C (Fig. 6I, 6J, 6K, and 6L). In Fig. 6E, unpolished aleurone layers remained at the dorsal surface of the grains with high fluorescence intensity (arrowheads). In magnified images, pullulanase-like immuno-reactivity was detected along endosperm cell walls and around starch granules at the dorsal (arrows in Fig. 6B, 6F, and 6J), central (arrows in Fig. 6C, 6G, and 6K), and ventral regions (arrows in Fig. 6D, 6H, and 6L) in the endosperm layers of Nipponbare. In comparison with Figure 2E and 2F, pullulanase-like immuno-reactivity around individual starch granules decreased in a temperature-dependent manner and that along endosperm cell walls was highly detected, especially in the central region of Nipponbare grains (Fig. 6C, 6G, and 6H). At the ventral region of grains, immuno-reactivity around starch granules was detected even when the temperature reached 60 °C (Fig. 6L).

Fig. 6.

Pullulanase-like immuno-reactivity was mainly detected along endosperm cell walls and partly around starch granules during the cooking process in Nipponbare cultivars. The longitudinal sections of polished rice grains were immunolabeled with monoclonal anti-pullulanase mouse IgG. Anti-mouse IgG conjugated with Alexa488 were used as a secondary antibody. The images for Nipponbare after the soaking step were shown at A (whole grains), B (dorsal region), C (central region), and D (ventral region) — those images when the temperature reached 40 °C shown at E, F, G, and H — those images when the temperature reached 60 °C shown at I, J, K, and L. Scale bars are 0.5 mm in A, E and I. In magnified images, pullulanase-like immuno-reactivity was detected along endosperm cell walls and around starch granules in the dorsal (arrows in B, F, and I), central (arrows in C, G, and K) and ventral (arrows in D, H, and L) endosperm layers. Arrows indicate representatives of positively stained areas. Scale bars are 40 µm.

Figure 7 shows images of pullulanase-like immuno-reactivity in Milkyqueen whole grains, and dorsal, central and ventral regions of grains after the soaking step (Fig. 7A, 7B, 7C, and 7D), when the temperature reached 40 °C (Fig. 7E, 7F, 7G, and 7H), and when the temperature reached 60 °C (Fig. 7I, 7J, 7K, and 7L). In magnified images, pullulanase-like immunoreactivity was detected along endosperm cell walls and around starch granules at the dorsal (arrows in Fig. 7B, 7F, and 7J), central (arrows in Fig. 7C, 7G, and 7K), and ventral parts (arrows in Fig. 7D, 7H, and 7L) in the endosperm layers of Milkyqueen. In comparison with Figure 2H and 2I, pullulanase-like immuno-reactivity around individual starch granules was decreased and the reactivity along endosperm cell walls was more intensely detected. At the ventral region of grains, immuno-reactivity around starch granules was detected even when the temperature reached 60 °C (Fig. 7L).

Fig. 7.

Pullulanase-like immuno-reactivity was detected along endosperm cell walls and around starch granules during the cooking process in Milkyqueen cultivars. The longitudinal sections of polished rice grains were immunolabeled with monoclonal anti-pullulanase mouse IgG. Anti-mouse IgG conjugated with Alexa488 were used as a secondary antibody. The images for Milkyqueen after the soaking step were shown at A (whole grains), B (dorsal region), C (central region), and D (ventral region) — those images when the temperature reached 40 °C shown at E, F, G, and H — those images when the temperature reached 60 °C shown at I, J, K, and L. Scale bars are 0.5 mm A, E and I. In magnified images, pullulanase-like immuno-reactivity was detected along endosperm cell walls and around starch granules in the dorsal (arrows in B, F, and I), central (arrows in C, G, and K) and ventral (arrows in D, H, and L) endosperm layers. In E, unpolished aleurone layers remained at the dorsal surface of the grains with high fluorescent intensity (arrowheads). Arrowheads indicate representatives of positively stained areas. Scale bars are 40 µm.

In Figures 5, 6, and 7, the fluorescence intensity of the central region of grains (C, G, and K) was less than that of the dorsal (B, F, and J) and ventral (D, H and L) regions. It has been reported that the amount of chemical components (reducing sugars, malto-oligosaccharides and amino acids) in the outer layer was greater than that in the inner layers of raw rice (Kishio and Aoyagi, 2014; Sugiyama et al., 1995; Tajima et al., 1992; Uyen Tran et al., 2004). The strong fluorescence intensity in the outer layer may explain the larger amounts of malto-oligosaccharides in the outer layer of rice grains. Quantitative analysis using ELISA for pullulanase among the outer and inner layers of rice grains is a future topic of research.

α-Glucosidase-like immuno-reactivity was predominant along endosperm cell walls of endosperm layers of non-glutinous polished rice during cooking    Making a longitudinal section of the polished rice grains as shown in Figure 1B, we analyzed α-glucosidase localization in polished rice grains during cooking of Koshihikari, Nipponbare and Milkyqueen. Figure 8 shows images of α-glucosidase-like immuno-reactivity in the posterior-dorsal regions of polished grains after the soaking step, when the temperature reached 40 °C, and when the temperature reached at 60 °C of Koshihikari (Fig. 8A, 8B, and 8C), Nipponbare (Fig. 8D, 8E, and 8F) and Milkyqueen (Fig. 8G, 8H, and 8I) cultivars. At each temperature, the immuno-reactivity was detected along the endosperm cell walls of each cultivar as shown in Fig.3A, 3B, 3C, 3D, 3E and 3F. In Fig. 8E, unpolished aleurone layers remained at the dorsal surface of the grains with high fluorescence intensity (arrowheads). These results strongly suggested that α-glucosidase was mainly localized along the endosperm cell walls and outside of amyloplasts in endosperm layers during the rice cooking process of Koshihikari, Nipponbare and Milkyqueen.

Fig. 8.

α-Glucosidase-like immuno-reactivity was detected along endosperm cell walls during the cooking process in non-glutinous polished rice. The longitudinal sections of brown rice grains were immunolabeled with the polyclonal anti-α- glucosidase rabbit IgG and anti-rabbit IgG conjugated with Alexa594 as a secondary antibody. The images for Koshihikari were shown at A(after the soaking step), B (at the temperature reached at 40 °C) , and C (at the temperature reached at 60 °C) — those of Nipponbare at D (after the soaking step), E(at the temperature reached at 40 °C), and F(at the temperature reached at 60 °C) — those of Milkyqueen at G (after the soaking step), H (at the temperature reached at 40 °C), and I (at the temperature reached at 60 °C). In E, unpolished aleurone layers remained at the dorsal surface of the grains with high fluorescent intensity (arrowheads). α-Glucosidase-like immuno-reactivity was detected along endosperm cell walls and outside of amyloplasts during the cooking process. Arrowheads indicate representatives of positively stained areas. Scale bars are 0.5 mm

In our previous report, α-glucosidase was released from rice grains to the cooking water in Koshihikari, but not in Nipponbare during the cooking process (Tsuyukubo et al., 2013). In this study, we found that α-glucosidase localization in Koshihikari and Nipponbare cultivars was mainly along endosperm cell walls of the endosperm layers during the cooking process (Fig. 8A, 8B, 8C, 8D, 8E, and 8F). The similar α-glucosidase localization in both cultivars raises the question of why the elution behavior of α-glucosidase differed in Koshihikari and Nipponbare cultivars. The diversity of cell-wall enzymes (Endo-1,4-β-glucanase) and their activities (β-xylanase, α-glucanase, and α-mannosidase) among various rice cultivars have been reported (Nakamura et al., 2012; Tsujii and Takano, 2015). Differences in the activity and localization of cell-wall degrading enzymes in rice endosperm layers may affect the elution behavior of α-glucosidase from Koshihikari and Nipponbare grains.

In this study, we revealed that α-glucosidase and pullulanase show distinct localizations in the endosperm layers of brown rice and cooked rice — each unique localization was common among the three non-glutinous rice cultivars. The distribution of each enzyme of other non-glutinous and glutinous cultivars is a future topic of research. Quantitative analysis with ELISA for enzymes using specific antibodies would lead to better understanding of how each enzyme contributes to glucose production during rice cooking. Taken together, the results in this study support the idea that the unique localization of each enzyme should be taken into account when discussing the mechanisms of starch degradation by enzymes during rice cooking.

Conclusion

In the three non-glutinous rice cultivars, pullulanase-like immuno-reactivity was mainly detected in amyloplasts and α-glucosidase-like immuno-reactivity was mainly found along endosperm cell walls in the endosperm layers of brown rice. Both immuno-reactivities overlapped in the aleurone layers of brown rice. During rice cooking, both immuno-reactivities were colocalized along endosperm cell walls after the soaking step, and when temperatures reached 40 °C and 60 °C. The common localization in the three non-glutinous cultivars indicated that enzyme localization plays an important role in the degradation of endosperm starch during rice cooking.

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